Morpholino modified oligomeric compounds

ABSTRACT

The present invention provides morpholino modified oligomeric compounds having at least one monomer subunit having Formula III, compounds having Formula I useful for making certain of the morpholino modified oligomeric compounds and methods of using the oligomeric compounds. In certain embodiments, the oligomeric compounds provided herein provide for an improved toxicity profile. Certain such oligomeric compounds are useful for hybridizing to a complementary nucleic acid, including but not limited, to nucleic acids in a cell. In certain embodiments, hybridization results in modulation of the amount of activity or expression of the target nucleic acid in a cell.

FIELD OF THE INVENTION

The present invention pertains generally to chemically-modifiedoligonucleotides for use in research, diagnostics, and/or therapeutics.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledCHEM0098WOSEQ_ST25.txt, created Feb. 9, 2018, which is 6 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

Antisense compounds have been used to modulate target nucleic acids.Antisense compounds comprising a variety of chemical modifications andmotifs have been reported. In certain instances, such compounds areuseful as research tools, diagnostic reagents, and as therapeuticagents. In certain instances, antisense compounds have been shown tomodulate protein expression by binding to a target messenger RNA (mRNA)encoding the protein. In certain instances, such binding of an antisensecompound to its target mRNA results in cleavage of the mRNA. Antisensecompounds that modulate processing of a pre-mRNA have also beenreported. Such antisense compounds alter splicing, interfere withpolyadenlyation or prevent formation of the 5′-cap of a pre-mRNA.

Generally, the principle behind antisense technology is that anantisense compound hybridizes to a target nucleic acid and modulatesgene expression activities or function, such as transcription ortranslation. The modulation of gene expression can be achieved by, forexample, target degradation or occupancy-based inhibition. An example ofmodulation of RNA target function by degradation is RNase H-baseddegradation of the target RNA upon hybridization with a DNA-likeantisense compound. Another example of modulation of gene expression bytarget degradation is RNA interference (RNAi). RNAi generally refers toantisense-mediated gene silencing involving the introduction of dsRNAleading to the sequence-specific reduction of targeted endogenous mRNAlevels. An additional example of modulation of RNA target function by anoccupancy-based mechanism is modulation of microRNA function. MicroRNAsare small non-coding RNAs that regulate the expression of protein-codingRNAs. The binding of an antisense compound to a microRNA prevents thatmicroRNA from binding to its messenger RNA targets, and thus interfereswith the function of the microRNA. Regardless of the specific mechanism,this sequence-specificity makes antisense compounds extremely attractiveas tools for target validation and gene functionalization, as well astherapeutics to selectively modulate the expression of genes involved inthe pathogenesis of malignancies and other diseases.

Antisense technology is an effective means for reducing the expressionof one or more specific gene products and can therefore prove to beuniquely useful in a number of therapeutic, diagnostic, and researchapplications. Chemically modified nucleosides are routinely used forincorporation into antisense compounds to enhance one or moreproperties, such as nuclease resistance, pharmacokinetics or affinityfor a target RNA. In 1998, the antisense compound, Vitravene®(fomivirsen; developed by Isis Pharmaceuticals Inc., Carlsbad, Calif.)was the first antisense drug to achieve marketing clearance from theU.S. Food and Drug Administration (FDA), and is currently a treatment ofcytomegalovirus (CMV)-induced retinitis in AIDS patients.

New chemical modifications have improved the potency and efficacy ofantisense compounds, uncovering the potential for oral delivery as wellas enhancing subcutaneous administration, decreasing potential for sideeffects, and leading to improvements in patient convenience. Chemicalmodifications increasing potency of antisense compounds allowadministration of lower doses, which reduces the potential for toxicity,as well as decreasing overall cost of therapy. Modifications increasingthe resistance to degradation result in slower clearance from the body,allowing for less frequent dosing. Different types of chemicalmodifications can be combined in one compound to further optimize thecompound's efficacy.

Targeting disease-causing gene sequences was first suggested more thanthirty years ago (Belikova et al., Tet. Lett. 1967, 8(37), 3557-3562),and antisense activity was demonstrated in cell culture more than adecade later (Zamecnik et al., Proc. Natl. Acad. Sci. U.S.A. 1978,75(1), 280-284). One advantage of antisense technology in the treatmentof a disease or condition that stems from a disease-causing gene is thatit is a direct genetic approach that has the ability to modulate(increase or decrease) the expression of specific disease-causing genes.Another advantage is that validation of a therapeutic target usingantisense compounds results in direct and immediate discovery of thedrug candidate; the antisense compound is the potential therapeuticagent.

Morpholino compounds and their use in oligomeric compounds has beenreported in numerous patents and published articles (see for example:Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos.5,698,685; 5,166,315; 5,185,444; and 5,034,506).

The synthesis and properties of morpholino chimeric oligonucleotides hasbeen previously reported (Zhang et al., Tetrahedron Letters, 2008, 49,3570-3573).

The interference in mammalian cells by siRNA's modified with morpholinonucleoside analogues has been previously reported (Zang et al.,Bioorganic & Medicinal Chemistry, 2009, 17, 2441-2446).

The synthesis of phosphorodiamidate morpholino oligonucleotides andtheir chimeras using phosphoramidite chemistry has been previouslyreported (Paul et al., J. A. C. S., 2016, 138(48), 15663-15672).

SUMMARY OF THE INVENTION

In certain embodiments, oligomeric compounds are provided having atleast one monomer subunit having Formula III, compounds having Formula Iuseful for making the oligomeric compounds and methods of using theoligomeric compounds.

In certain embodiments, compounds are provided having Formula I:

wherein:

Bx is an optionally protected heterocyclic base moiety;

T₁ is H or a hydroxyl protecting group; and

R₁ is C₁₋₆ alkyl or substituted C₁₋₆ alkyl comprising an optionallyprotected substituent group selected from halogen, OJ₁, NJ₁J₂, SJ₁, N₃,OC(=Q)J₁, OC(=Q)NJ₁J₂, NJ₃C(=Q)NJ₁J₂ and CN, wherein each J₁, J₂ and J₃is, independently, H or C₁-C₆ alkyl, and Q is O, S or NJ₁.

In certain embodiments, Bx is uracil, thymine, cytosine,4-N-benzoylcytosine, 4-N-isobutyrylcytosine, 5′-methyl cytosine,5′-methyl-4-N-benzoylcytosine, 5′-methyl-4-N-isbutyrylcytosine, adenine,6-N-benzoyladenine, guanine or 2-N-isobutyrylguanine. In certainembodiments, Bx is uracil, thymine, 5′-methyl-4-N-isbutyrylcytosine,6-N-benzoyladenine or 2-N-isobutyrylguanine.

In certain embodiments, T₁ is 4,4′-dimethoxytrityl.

In certain embodiments, R₁ is C₁₋₆ alkyl. In certain embodiments, R₁ ismethyl. In certain embodiments, R₁ is a substituted C₁₋₆ alkyl. Incertain embodiments, the substituted C₁₋₆ alkyl is substituted with OJ₁wherein J₁ is C₁-C₆ alkyl. In certain embodiments, R₁ is —(CH₂)₃—OCH₃.

In certain embodiments, oligomeric compounds are provided comprisingfrom 10 to 30 monomer subunits wherein at least one monomer subunit hasFormula III:

wherein independently for each monomer subunit having Formula III:

Bx is an optionally protected heterocyclic base moiety;

T₃ is hydroxyl, a protected hydroxyl, an internucleoside linking grouplinking the monomer subunit of Formula III to the 5′-remainder of theoligomeric compound or a terminal group;

T₅ is H, C₁₋₆ alkyl, a terminal group or a nitrogen protecting groupwhen located on the 3′-terminus of the oligomeric compound wherein atleast one T₅ is a linking group having the formula:

wherein independently for each T₅:

R₂ is hydroxyl, C₁₋₆ alkyl or substituted C₁₋₆ alkyl comprising anoptionally protected substituent group selected from halogen, OJ₁,NJ₁J₂, SJ₁, N₃, OC(=Q)J₁, OC(=Q)NJ₁J₂, NJ₃C(=Q)NJ₁J₂ and CN, whereineach J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and Q is O, S orNJ₁;

X is O or S wherein when X is O, at least one R₂ is other than hydroxyl;and

T₄ is a single bond connecting said linking group to the 3′-remainder ofthe oligomeric compound or T₄ forms a single bond with a T₃ from asecond monomer subunit having Formula III.

In certain embodiments, each Bx is, independently, an optionallyprotected pyrimidine, substituted pyrimidine, purine or substitutedpurine. In certain embodiments, each Bx is, independently, uracil,thymine, cytosine, 4-N-benzoylcytosine, 4-N-isobutyrylcytosine,5′-methyl cytosine, 5′-methyl-4-N-benzoylcytosine,5′-methyl-4-N-isbutyrylcytosine, adenine, 6-N-benzoyladenine, guanine or2-N-isobutyrylguanine. In certain embodiments, each Bx is,independently, uracil, thymine, cytosine, 5-methylcytosine, adenine orguanine.

In certain embodiments, at least one T₃ is an internucleoside linkinggroup linking the monomer subunit of Formula III to the 5′-remainder ofthe oligomeric compound. In certain embodiments, one T₃ is hydroxyl. Incertain embodiments, one T₃ is a terminal group.

In certain embodiments, one T₅ is H, C₁-C₆ alkyl or a nitrogenprotecting group. In certain embodiments, one T₅ is a methyl. In certainembodiments, one T₅ is 4,4′-dimethoxytrityl. In certain embodiments, oneT₅ is a terminal group. In certain embodiments, one T₅ is said linkinggroup. In certain embodiments, each T₅ is said linking group.

In certain embodiments, at least one T₄ is a single bond connecting saidlinking group to the 3′-remainder of the oligomeric compound. In certainembodiments, at least one T₄ forms a single bond with a T₃ from a secondmonomer subunit having Formula III.

In certain embodiments, the 5′-remainder of the oligomeric compoundcomprises a 3′-terminal 3-D-2′-deoxyribonucleoside. In certainembodiments, the 3′-remainder of the oligomeric compound comprises a5′-terminal 3-D-2′-deoxyribonucleoside. In certain embodiments, at leastone of the 5′-terminal monomer and the 3′-terminal monomer of theoligomeric compound comprises a terminal group.

In certain embodiments, each X is O. In certain embodiments, each X isS.

In certain embodiments, each R₂ is hydroxyl. In certain embodiments,each R₂ is C₁₋₆ alkyl. In certain embodiments, each R₂ is methyl. Incertain embodiments, each R₂ is substituted C₁₋₆ alkyl. In certainembodiments, the substituent group is OJ₁. In certain embodiments, J₁ isC₁-C₆ alkyl. In certain embodiments, J₁ is methyl. In certainembodiments, each R₂ is —(CH₂)₃—OCH₃.

In certain embodiments, essentially each monomer subunit has FormulaIII.

In certain embodiments, oligomeric compounds are provided comprising agapped oligomeric compound having a first region consisting of from 2 to5 monomer subunits, a second region consisting of from 2 to 5 monomersubunits and a gap region located between the first and second regionconsisting of from 8 to 12 monomer subunits wherein each monomer subunitin the first and second region is a modified nucleoside and each monomersubunit in the gap region is an unmodified nucleoside or a modifiednucleoside different from the modified nucleosides in the first andsecond region and wherein at least one of the monomer subunits hasFormula III. In certain embodiments, each monomer subunit in the gapregion other than monomer subunits having Formula III is aβ-D-2′-deoxyribonucleoside. In certain embodiments, each monomer subunitin the gap region is a β-D-2′-deoxyribonucleoside.

In certain embodiments, one monomer subunit having Formula III in thegap region. In certain embodiments, two monomer subunits having FormulaIII in the gap region. In certain embodiments, three monomer subunitshaving Formula III in the gap region.

In certain embodiments, at least one monomer subunit having Formula IIIin one of the first and second regions. In certain embodiments, at twomonomer subunits having Formula III in the first and second regions. Incertain embodiments, 3 to 4 monomer subunits having Formula III in thefirst and second regions.

In certain embodiments, the gap region has from about 10 to about 12monomer subunits. In certain embodiments, the gap region has 10 monomersubunits.

In certain embodiments, the first and second regions each comprise 2monomer subunits. In certain embodiments, the first and second regionseach comprise 3 monomer subunits. In certain embodiments, the first andsecond regions each comprise 5 monomer subunits. In certain embodiments,each modified nucleoside in the first and second region comprises amodified sugar moiety. In certain embodiments, each modified nucleosidein the first and second region comprises the same modified sugar moiety.

In certain embodiments, at least two modified nucleosides in the firstand second region comprises differently modified sugar moieties. Incertain embodiments, each modified nucleoside in the first and secondregion is, independently, a bicyclic nucleoside comprising a bicyclicfuranosyl sugar moiety or a modified nucleoside comprising a furanosylsugar moiety having at least one substituent group. In certainembodiments, each modified nucleoside in the first and second region is,independently, a bicyclic nucleoside comprising a 4′-CH((S)—CH₃)—O-2′bridge or a 2′-O-methoxyethyl substituted nucleoside. In certainembodiments, at least one modified nucleoside in the first and secondregion comprises a sugar surrogate.

In certain embodiments, each internucleoside linking group other than alinking group (T₅) is, independently, a phosphodiester internucleosidelinking group or a phosphorothioate internucleoside linking group. Incertain embodiments, essentially each internucleoside linking groupother than a linking group (T₅) is a phosphorothioate internucleosidelinking group.

In certain embodiments, oligomeric compounds are provided comprisingfrom 12 to 28 monomer subunits comprising at least oneT(Z_(a)T_(b))_(c)Z_(d) motif where each a and b is, independently, from1 to about 8, c is from 1 to about 12, d is 0 or 1, one of T and Z is aβ-D-2′-deoxyribonucleoside and the other of T and Z is a monomer subunithaving Formula III:

wherein independently for each monomer subunit having Formula III:

Bx is an optionally protected heterocyclic base moiety;

T₃ is hydroxyl, a protected hydroxyl, an internucleoside linking grouplinking the monomer subunit of Formula III to the 5′-remainder of theoligomeric compound or a terminal group;

T₅ is H, C₁₋₆ alkyl, a nitrogen protecting or a linking group having theformula:

wherein independently for each T₅:

R₂ is hydroxyl, C₁₋₆ alkyl or substituted C₁₋₆ alkyl comprising anoptionally protected substituent group selected from halogen, OJ₁,NJ₁J₂, SJ₁, N₃, OC(=Q)J₁, OC(=Q)NJ₁J₂, NJ₃C(=Q)NJ₁J₂ and CN, whereineach J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and Q is O, S orNJ₁;

X is O or S; and

T₄ is a single bond connecting said linking group to the 3′-remainder ofthe oligomeric compound or T₄ forms a single bond with a T₃ from asecond monomer subunit having Formula III.

In certain embodiments, each Bx is, independently, an optionallyprotected pyrimidine, substituted pyrimidine, purine or substitutedpurine. In certain embodiments, each Bx is, independently, uracil,thymine, cytosine, 5-methylcytosine, adenine or guanine.

In certain embodiments, each X is O. In certain embodiments, each X isS.

In certain embodiments, each R₂ is hydroxyl. In certain embodiments,each R₂ is C₁₋₆ alkyl. In certain embodiments, each R₂ is methyl. Incertain embodiments, each R₂ is substituted C₁₋₆ alkyl.

In certain embodiments, the substituent group is OJ₁. In certainembodiments, J₁ is C₁-C₆ alkyl. In certain embodiments, J₁ is methyl. Incertain embodiments, each R₂ is —(CH₂)₃—OCH₃.

In certain embodiments, oligomeric compounds are provided comprising oneT(Z_(a)T_(b))_(c)Z_(d) motif. In certain embodiments, oligomericcompounds are provided comprising two T(Z_(a)T_(b))_(c)Z_(d) motifsseparated by a central region of from 2 to 14 nucleosides. In certainembodiments, oligomeric compounds are provided comprising at least threeT(Z_(a)T_(b))_(c)Z_(d) motifs wherein each T(Z_(a)T_(b))_(c)Z_(d) motifis separated by at least 1 nucleoside.

In certain embodiments, each a and b is 1. In certain embodiments, eacha and b is, independently, from 1 to 3. In certain embodiments, each aand b is, independently, from 1 to 5. In certain embodiments, each a andb is, independently, from 1 to 7.

In certain embodiments, c is 1. In certain embodiments, c is 2. Incertain embodiments, c is 3. In certain embodiments, c is from 4 to 6.

In certain embodiments, d is 0. In certain embodiments, d is 1.

In certain embodiments, T is a β-D-2′-deoxyribonucleoside.

In certain embodiments, Z is a β-D-2′-deoxyribonucleoside.

In certain embodiments, each internucleoside linking group other than alinking group (T₅) is, independently, a phosphodiester internucleosidelinking group or a phosphorothioate internucleoside linking group. Incertain embodiments, each internucleoside linking group other than alinking group (T₅) is a phosphorothioate internucleoside linking group.

In certain embodiments, the oligomeric compound comprises at least from1 to 3 3′-terminal β-D-2′-deoxyribonucleoside. In certain embodiments,the oligomeric compound comprises at least from 1 to 3 5′-terminalβ-D-2′-deoxyribonucleosides. In certain embodiments, the oligomericcompound comprises a 5′ or 3′-terminal group.

In certain embodiments, the oligomeric compound comprises from 12 to 22monomer subunits. In certain embodiments, the oligomeric compoundcomprises from 14 to 20 monomer subunits. In certain embodiments, theoligomeric compound comprises from 14 to 16 monomer subunits. In certainembodiments, the oligomeric compound comprises from 16 to 20 monomersubunits.

In certain embodiments, oligomeric compounds are provided comprisingfrom 10 to 30 monomer subunits wherein at least one monomer subunit hasFormula II:

wherein independently for each monomer subunit having Formula II:

Bx is an optionally protected heterocyclic base moiety;

T₃ is H, a hydroxyl protecting group, an internucleoside linking grouplinking the monomer subunit of Formula II to the 5′-remainder of theoligomeric compound or a terminal group;

T₄ is a single bond connecting the monomer subunit having Formula II tothe 3′-remainder of the oligomeric compound;

R₂ is HO, C₁₋₆ alkyl or substituted C₁₋₆ alkyl comprising an optionallyprotected substituent group selected from halogen, OJ₁, NJ₁J₂, SJ₁, N₃,OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ and J₃is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁; and

X is O or S wherein when X is O, R₂ is other than OH.

In certain embodiments, oligomeric compounds are provided comprisingfrom 10 to 30 monomer subunits wherein at least one monomer subunit hasFormula II:

wherein independently for each monomer subunit having Formula II:

Bx is an optionally protected heterocyclic base moiety;

T₃ is H, a hydroxyl protecting group, an internucleoside linking grouplinking the monomer subunit of Formula II to the 5′-remainder of theoligomeric compound or a terminal group;

T₄ is a single bond connecting the monomer subunit having Formula II tothe 3′-remainder of the oligomeric compound;

R₂ is HO, C₁₋₆ alkyl or substituted C₁₋₆ alkyl comprising an optionallyprotected substituent group selected from halogen, OJ₁, NJ₁J₂, SJ₁, N₃,OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ and J₃is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁; and

X is O or S wherein when X is O and R₂ is OH then the oligomericcompound is other than an siRNA oligomeric compound.

In certain embodiments, each Bx is, independently, an optionallyprotected pyrimidine, substituted pyrimidine, purine or substitutedpurine. In certain embodiments, Bx is, independently, uracil, thymine,cytosine, 5-methylcytosine, adenine or guanine.

In certain embodiments, at least one T₃ is an internucleoside linkinggroup linking the monomer subunit of Formula II to the 5′-remainder ofthe oligomeric compound.

In certain embodiments, the 5′-remainder of the oligomeric compoundcomprises a 3′-terminal β-D-2′-deoxyribonucleoside.

In certain embodiments, one T₃ is H. In certain embodiments, one T₃ is aterminal group.

In certain embodiments, the 3′-remainder of the oligomeric compoundcomprises a 5′-terminal β-D-2′-deoxyribonucleoside. In certainembodiments, the 5′-terminal monomer of the 5′-remainder of theoligomeric compound or the 3′-terminal monomer of the 3′-remainder ofthe oligomeric compound comprises a terminal group.

In certain embodiments, each X is O. In certain embodiments, each X isS.

In certain embodiments, each R₂ is OH. In certain embodiments, each R₂is C₁₋₆ alkyl. In certain embodiments, each R₂ is methyl. In certainembodiments, each R₂ is substituted C₁₋₆ alkyl. In certain embodiments,the substituent group is OJ₁. In certain embodiments, J₁ is C₁-C₆ alkyl.In certain embodiments, J₁ is methyl. In certain embodiments, each R₂ is—(CH₂)₃—OCH₃.

In certain embodiments, essentially each monomer subunit has Formula II.

In certain embodiments, the oligomeric compound comprises a gappedoligomeric compound having a first region consisting of from 2 to 5monomer subunits, a second region consisting of from 2 to 5 monomersubunits and a gap region located between the first and second regionconsisting of from 8 to 12 monomer subunits wherein each monomer subunitin the first and second region is a modified nucleoside and each monomersubunit in the gap region is an unmodified nucleoside or a modifiednucleoside different from the modified nucleosides in the first andsecond region and wherein at least one of the monomer subunits hasFormula II.

In certain embodiments, gapped oligomeric compounds are provided whereinmonomer subunits of Formula II are located at one or more positions thatdon't include a gap junction.

In certain embodiments, each monomer subunit in the gap region otherthan a monomer subunit having Formula II is aβ-D-2′-deoxyribonucleoside. In certain embodiments, each monomer subunitin the gap region is a β-D-2′-deoxyribonucleoside.

In certain embodiments, the oligomeric compound comprises one monomerhaving Formula II in the gap region. In certain embodiments, theoligomeric compound comprises two monomers having Formula II in the gapregion. In certain embodiments, the oligomeric compound comprises threemonomers having Formula II in the gap region.

In certain embodiments, the oligomeric compound comprises at least onemonomer having Formula II in one of the first and second regions. Incertain embodiments, the oligomeric compound comprises at least twomonomers having Formula II in the first and second regions. In certainembodiments, the oligomeric compound comprises from 3 to 4 monomershaving Formula II in the first and second regions.

In certain embodiments, the gap region has from about 10 to about 12monomer subunits. In certain embodiments, the gap region has 10 monomersubunits.

In certain embodiments, the first and second regions each comprise 2monomer subunits. In certain embodiments, the first and second regionseach comprise 3 monomer subunits. In certain embodiments, the first andsecond regions each comprise 5 monomer subunits. In certain embodiments,each modified nucleoside in the first and second region comprises amodified sugar moiety. In certain embodiments, each modified nucleosidein the first and second region comprises the same type of modified sugarmoiety. In certain embodiments, at least two modified nucleosides in thefirst and second region comprises differently modified sugar moieties.In certain embodiments, each modified nucleoside in the first and secondregion is, independently, a bicyclic nucleoside comprising a bicyclicfuranosyl sugar moiety or a modified nucleoside comprising a furanosylsugar moiety having at least one substituent group. In certainembodiments, each modified nucleoside in the first and second region is,independently, a bicyclic nucleoside comprising a 4′-CH((S)—CH₃)—O-2′bridge or a 2′-O-methoxyethyl substituted nucleoside. In certainembodiments, at least one modified nucleoside in the first and secondregion comprises a sugar surrogate.

In certain embodiments, each internucleoside linking group other than alinking group included in a monomer subunit having Formula II is,independently, a phosphodiester internucleoside linking group or aphosphorothioate internucleoside linking group. In certain embodiments,essentially each internucleoside linking group other than a linkinggroup included in a monomer subunit having Formula II is aphosphorothioate internucleoside linking group.

In certain embodiments, methods of inhibiting gene expression areprovided comprising contacting one or more cells, a tissue or an animalwith an oligomeric compound as disclosed herein that is complementary toa target RNA.

In certain embodiments, the cells are in a human. In certainembodiments, the target RNA is human mRNA. In certain embodiments, thetarget RNA is cleaved thereby inhibiting its function.

In certain embodiments, an in vitro method of inhibiting gene expressionis provided comprising contacting one or more cells or a tissue with anoligomeric compound as provided herein.

In certain embodiments, oligomeric compounds are provided for use in anin vivo method of inhibiting gene expression comprising contacting oneor more cells, a tissue or an animal with an oligomeric compound of anyof claims 10 to 97.

In certain embodiments, oligomeric compounds are provided for use inmedical therapy.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are oligomeric compounds having at least one monomersubunit having Formula III, compounds having Formula I useful for makingcertain of the oligomeric compounds and methods of using the oligomericcompounds. In certain embodiments, the oligomeric compounds providedherein are useful for modulating the activity of a target nucleic acid.In certain embodiments, the morpholino modified oligomeric compoundsprovided herein provide an improved toxicity profile compared to anotherwise unmodified oligomeric compound.

In certain embodiments, oligomeric compounds are provided having atleast one monomer subunit having Formula III, compounds having Formula Iuseful for making the oligomeric compounds and methods of using theoligomeric compounds.

In certain embodiments, oligomeric compounds are provided comprisingfrom 10 to 30 monomer subunits wherein at least one monomer subunit hasFormula III:

wherein independently for each monomer subunit having Formula III:

Bx is an optionally protected heterocyclic base moiety;

T₃ is hydroxyl, a protected hydroxyl, an internucleoside linking grouplinking the monomer subunit of Formula III to the 5′-remainder of theoligomeric compound or a terminal group;

T₅ is H, C₁₋₆ alkyl, a terminal group or a nitrogen protecting groupwhen located on the 3′-terminus of the oligomeric compound wherein atleast one T₅ is a linking group having the formula:

wherein independently for each T₅:

R₂ is hydroxyl, C₁₋₆ alkyl or substituted C₁₋₆ alkyl comprising anoptionally protected substituent group selected from halogen, OJ₁,NJ₁J₂, SJ₁, N₃, OC(=Q)J₁, OC(=Q)NJ₁J₂, NJ₃C(=Q)NJ₁J₂ and CN, whereineach J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and Q is O, S orNJ₁;

X is O or S wherein when X is O, at least one R₂ is other than hydroxyl;and

T₄ is a single bond connecting said linking group to the 3′-remainder ofthe oligomeric compound or T₄ forms a single bond with a T₃ from asecond monomer subunit having Formula III

In certain embodiments, X is O and R₂ is OH and the oligomeric compoundis other than an siRNA.

In certain embodiments, gapped oligomeric compounds are providedconsisting of from 2 to 5 monomer subunits, a second region consistingof from 2 to 5 monomer subunits and a gap region located between thefirst and second region consisting of from 8 to 12 monomer subunitswherein each monomer subunit in the first and second region is amodified nucleoside and each monomer subunit in the gap region is anunmodified nucleoside or a modified nucleoside different from themodified nucleosides in the first and second region and wherein at leastone of the monomer subunits has Formula II.

In certain embodiments, gapped oligomeric compounds as provided hereinare described by the shorthand E₅/G/E₃ wherein the “E₅” is the externalregion at the 5′-end, “G” is the gap region and “E₃” is the externalregion at the 3′-end. In certain embodiments, gapped oligomericcompounds are provided comprising a 2/10/2 motif. In certainembodiments, gapped oligomeric compounds are provided comprising a3/10/3 motif. In certain embodiments, gapped oligomeric compounds areprovided comprising a 5/10/5 motif. In certain embodiments, the modifiednucleosides in the external regions are bicyclic modified nucleosides.In certain embodiments, the modified nucleosides in the external regionseach comprise a 2′-substituent group selected from F, OCH₃, O(CH₂)₂—OCH₃and OCH₂C(═O)—N(H)CH₃. In certain embodiments, the modified nucleosidesin the external regions are a mixture of bicyclic modified nucleosidesand modified nucleosides comprising at least one substituent group. Incertain embodiments, the modified nucleosides in the external regionsare a mixture of bicyclic modified nucleosides comprising a bridginggroup selected from 4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-CH(CH₃)—O-2′,4′-CH₂—N(CH₃)—O-2′, 4′-CH₂—C(H)(CH₃)-2′ and 4′-CH₂—C(═CH₂)-2′ andmodified nucleosides comprising a 2′-substituent group selected from F,OCH₃, O(CH₂)₂—OCH₃ and OCH₂C(═O)—N(H)CH₃. In certain embodiments, themodified nucleosides in the external regions are a mixture of bicyclicmodified nucleosides comprising a bridging group selected from4′-CH₂—O-2′ or 4′-CH[(S)—(CH₃)]—O-2′ and modified nucleosides comprisinga 2′-substituent group selected from F, OCH₃, O(CH₂)₂—OCH₃ andOCH₂C(═O)—N(H)CH₃. In certain embodiments, the modified nucleosides inthe external regions are a mixture of bicyclic modified nucleosidescomprising a bridging group selected from 4′-CH[(S)—(CH₃)]—O-2′ andmodified nucleosides comprising a 2′-O(CH₂)₂—OCH₃ substituent group. Incertain embodiments, each modified nucleoside in each external region isa bicyclic modified nucleoside comprising a 4′-CH[(S)—(CH₃)]—O-2′bridging group. In certain embodiments, each modified nucleoside in eachexternal region is a 2′-O(CH₂)₂—OCH₃ modified nucleoside. In certainembodiments, the gapped oligomeric compound is further functionalized byaddition of a conjugate group.

In certain embodiments, oligomeric compounds are provided comprisingfrom 12 to 28 monomer subunits comprising at least oneT(Z_(a)T_(b))_(c)Z_(d) motif where each a and b is, independently, from1 to about 8, c is from 1 to about 12, d is 0 or 1, one of T and Z is aβ-D-2′-deoxyribonucleo-side and the other of T and Z is a monomersubunit having Formula III:

wherein independently for each monomer subunit having Formula III:

Bx is an optionally protected heterocyclic base moiety;

T₃ is hydroxyl, a protected hydroxyl, an internucleoside linking grouplinking the monomer subunit of Formula III to the 5′-remainder of theoligomeric compound or a terminal group;

T₅ is H, C₁₋₆ alkyl, a nitrogen protecting or a linking group having theformula:

wherein independently for each T₅:

R₂ is hydroxyl, C₁₋₆ alkyl or substituted C₁₋₆ alkyl comprising anoptionally protected substituent group selected from halogen, OJ₁,NJ₁J₂, SJ₁, N₃, OC(=Q)J₁, OC(=Q)NJ₁J₂, NJ₃C(=Q)NJ₁J₂ and CN, whereineach J₁, J₂ and J₃ is, independently, H or C₁-C₆ alkyl, and Q is O, S orNJ₁;

X is O or S; and

T₄ is a single bond connecting said linking group to the 3′-remainder ofthe oligomeric compound or T₄ forms a single bond with a T₃ from asecond monomer subunit having Formula III.

In certain embodiments, compounds having reactive phosphorus groups areprovided that are useful for preparing oligomeric compounds of theinvention on an automated synthesizer. In certain embodiments, compoundshaving reactive phosphorus groups are provided having Formula I:

wherein:

Bx is an optionally modified heterocyclic base moiety;

T₁ is H or a hydroxyl protecting group; and

R₁ is C₁₋₆ alkyl or substituted C₁₋₆ alkyl comprising an optionallyprotected substituent group selected from halogen, OJ₁, NJ₁J₂, SJ₁, N₃,OC(═X)J₁, OC(═X)NJ₁J₂, NJ₃C(═X)NJ₁J₂ and CN, wherein each J₁, J₂ and J₃is, independently, H or C₁-C₆ alkyl, and X is O, S or NJ₁. In certainembodiments, R₁ is alkyl such as methyl. In certain embodiments, R₁ issubstituted alkyl such as alkoxy substituted alkyl. In certainembodiments, R₁ is CH₃O(CH₂)₃—.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive. Herein, the use of the singular includes theplural unless specifically stated otherwise. As used herein, the use of“or” means “and/or” unless stated otherwise. Furthermore, the use of theterm “including” as well as other forms, such as “includes” and“included”, is not limiting. Also, terms such as “element” or“component” encompass both elements and components comprising one unitand elements and components that comprise more than one subunit, unlessspecifically stated otherwise.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including, but not limited to, patents, patent applications, articles,books, and treatises, are hereby expressly incorporated-by-reference forthe portions of the document discussed herein, as well as in theirentirety.

Definitions

Unless specific definitions are provided, the nomenclature used inconnection with, and the procedures and techniques of, analyticalchemistry, synthetic organic chemistry, and medicinal and pharmaceuticalchemistry described herein are those well-known and commonly used in theart. Where permitted, all patents, applications, published applicationsand other publications and other data referred to throughout in thedisclosure are incorporated by reference herein in their entirety.

Unless otherwise indicated, the following terms have the followingmeanings:

As used herein, “2′-deoxynucleoside” means a nucleoside comprising2′-H(H) furanosyl sugar moiety, as found in naturally occurringdeoxyribonucleic acids such as a β-D-2′-deoxyribnucleoside (DNA). Incertain embodiments, a 2′-deoxynucleoside may comprise a modifiednucleobase or may comprise an RNA nucleobase (uracil).

As used herein, “2′-substituted nucleoside” or “2-modified nucleoside”means a nucleoside comprising a 2′-substituted or 2′-modified sugarmoiety. As used herein, “2′-substituted” or “2-modified” in reference toa sugar moiety means a sugar moiety comprising at least one2′-substituent group other than H or OH.

As used herein, “Antisense activity” means any detectable and/ormeasurable change attributable to the hybridization of an antisensecompound to its target nucleic acid. In certain embodiments, antisenseactivity is a decrease in the amount or expression of a target nucleicacid or protein encoded by such target nucleic acid compared to targetnucleic acid levels or target protein levels in the absence of theantisense compound. In certain embodiments, antisense activity is achange in splicing of a pre-mRNA nucleic acid target. In certainembodiments, antisense activity is an increase in the amount orexpression of a target nucleic acid or protein encoded by such targetnucleic acid compared to target nucleic acid levels or target proteinlevels in the absence of the antisense compound.

As used herein, “Antisense compound” means a compound comprising anantisense oligonucleotide and optionally one or more additionalfeatures, such as a conjugate group or terminal group.

As used herein, “Antisense oligonucleotide” means an oligonucleotidethat (1) has a nucleobase sequence that is at least partiallycomplementary to a target nucleic acid and that (2) is capable ofproducing an antisense activity in a cell or animal.

As used herein, “Ameliorate” in reference to a treatment meansimprovement in at least one symptom relative to the same symptom in theabsence of the treatment. In certain embodiments, amelioration is thereduction in the severity or frequency of a symptom or the delayed onsetor slowing of progression in the severity or frequency of a symptom.

As used herein, “Bicyclic nucleoside” or “BNA” means a nucleosidecomprising a bicyclic sugar moiety. As used herein, “bicyclic sugar” or“bicyclic sugar moiety” means a modified sugar moiety comprising tworings, wherein the second ring is formed via a bridge or bridging groupconnecting two of the atoms in the first ring thereby forming a bicyclicstructure. In certain embodiments, the first ring of the bicyclic sugarmoiety is a furanosyl moiety. In certain embodiments, the bicyclic sugarmoiety does not comprise a furanosyl moiety.

As used herein, “Branching group” means a group of atoms having at least3 positions that are capable of forming covalent linkages to at least 3groups. In certain embodiments, a branching group provides a pluralityof reactive sites for connecting tethered ligands to an oligonucleotidevia a conjugate linker and/or a cleavable moiety.

As used herein, “Cell-targeting moiety” means a conjugate group orportion of a conjugate group that is capable of binding to a particularcell type or particular cell types.

As used herein, “Cleavable moiety” means a bond or group of atoms thatis cleaved under physiological conditions, for example, inside a cell,an animal, or a human.

As used herein, “Complementary” in reference to an oligonucleotide meansthat at least 70% of the nucleobases of such oligonucleotide or one ormore regions thereof and the nucleobases of another nucleic acid or oneor more regions thereof are capable of hydrogen bonding with one anotherwhen the nucleobase sequence of the oligonucleotide and the othernucleic acid are aligned in opposing directions. Complementarynucleobases means nucleobases that are capable of forming hydrogen bondswith one another. Complementary nucleobase pairs include, but unlessotherwise specific are not limited to, adenine (A) and thymine (T),adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methylcytosine (^(m)C) and guanine (G). Complementary oligonucleotides and/ornucleic acids need not have nucleobase complementarity at eachnucleoside. Rather, some mismatches are tolerated. As used herein,“fully complementary” or “100% complementary” in reference tooligonucleotides means that such oligonucleotides are complementary toanother oligonucleotide or nucleic acid at each nucleoside of theoligonucleotide.

As used herein, “Conjugate group” means a group of atoms that isdirectly or indirectly attached to an oligonucleotide. Conjugate groupsinclude a conjugate group and a conjugate linker that attaches theconjugate group to the oligonucleotide wherein the attachment mayinclude a cleavable moiety.

As used herein, “Conjugate linker” means a group of atoms comprising atleast one bond that connects a conjugate group to an oligonucleotidewherein the attachment may include a cleavable moiety.

As used herein, “Contiguous” in the context of an oligonucleotide refersto nucleosides, nucleobases, sugar moieties, or internucleoside linkagesthat are immediately adjacent to each other. For example, “contiguousnucleobases” means nucleobases that are immediately adjacent to eachother in a sequence.

As used herein, “Duplex” means two oligomeric compounds that are paired.In certain embodiments, the two oligomeric compounds are paired viahybridization of complementary nucleobases.

As used herein, “Extra-hepatic cell type” means a cell type that is nota hepatocyte.

As used herein, “Extra-hepatic nucleic acid target” means a targetnucleic acid that is expressed in tissues other than liver. In certainembodiments, extra-hepatic nucleic acid targets are not expressed in theliver or not expressed in the liver at a significant level. In certainembodiments, extra-hepatic nucleic acid targets are expressed outsidethe liver and also in the liver.

As used herein, “Extra-hepatic tissue” means a tissue other than liver.

As used herein, “Fully modified” in reference to a modifiedoligonucleotide means a modified oligonucleotide in which each sugarmoiety is modified. “Uniformly modified” in reference to a modifiedoligonucleotide means a fully modified oligonucleotide in which eachsugar moiety is the same. For example, the nucleosides of a uniformlymodified oligonucleotide can each have a 2′-MOE modification butdifferent nucleobase modifications, and the internucleoside linkages maybe different.

As used herein, “Gapmer” means an antisense oligonucleotide comprisingan internal region having a plurality of nucleosides that support RNaseH cleavage positioned between external regions having one or morenucleosides, wherein the nucleosides comprising the internal region arechemically distinct from the nucleoside or nucleosides comprising theexternal regions. The internal region may be referred to as the “gap”and the external regions may be referred to as the “wings.”

As used herein, “Hybridization” means the pairing or annealing ofcomplementary oligonucleotides and/or nucleic acids. While not limitedto a particular mechanism, the most common mechanism of hybridizationinvolves hydrogen bonding, which may be Watson-Crick, Hoogsteen orreversed Hoogsteen hydrogen bonding, between complementary nucleobases.

As used herein, “Inhibiting the expression or activity” refers to areduction or blockade of the expression or activity relative to theexpression of activity in an untreated or control sample and does notnecessarily indicate a total elimination of expression or activity.

As used herein, “Internucleoside linkage” or “internucleoside linkinggroup” means a group or bond that forms a covalent linkage betweenadjacent nucleosides in an oligonucleotide. As used herein “modifiedinternucleoside linkage” means any internucleoside linkage other than anaturally occurring, phosphate internucleoside linkage.Non-phosphodiester linkages are referred to herein as modifiedinternucleoside linkages. “Phosphorothioate linkage” means a modifiedphosphodiester linkage in which one of the non-bridging oxygen atoms isreplaced with a sulfur atom. A phosphorothioate internucleoside linkageis a modified internucleoside linkage. Modified internucleoside linkagesinclude linkages that comprise abasic nucleosides. As used herein,“abasic nucleoside” means a sugar moiety in an oligonucleotide oroligomeric compound that is not directly connected to a nucleobase. Incertain embodiments, an abasic nucleoside is adjacent to one or twonucleosides in an oligonucleotide.

As used herein, “Lipophilic group” or “lipophilic” in reference to achemical group means a group of atoms that is more soluble in lipids ororganic solvents than in water and/or has a higher affinity for lipidsthan for water. In certain embodiments, lipophilic groups comprise alipid. As used herein “lipid” means a molecule that is not soluble inwater or is less soluble in water than in organic solvents. In certainembodiments, compounds of the present invention comprise lipids selectedfrom saturated or unsaturated fatty acids, steroids, fat solublevitamins, phospholipids, sphingolipids, hydrocarbons, mono-, di-, andtri-glycerides, and synthetic derivatives thereof.

As used herein the term “monomer subunit” is meant to include all mannerof monomers that are amenable to oligomer synthesis. In general amonomer subunit includes at least a sugar moiety or modified sugarmoiety having at least two reactive sites that can form linkages tofurther monomer subunits. Essentially all monomer subunits include anucleobase that is hybridizable to a complementary site on a nucleicacid target. Reactive sites on monomer subunits located on the terminiof an oligomeric compound can be protected or unprotected (generally OH)or can form an attachment to a terminal group (conjugate or othergroup). Monomer subunits include, without limitation, nucleosides andmodified nucleosides. In certain embodiments, monomer subunits includenucleosides such as β-D-ribonucleosides and β-D-2′-deoxyribnucleosidesand modified nucleosides including but not limited to substitutednucleosides (such as 2′, 5′ and bis substituted nucleosides),4′-S-modified nucleosides (such as 4′-S-ribonucleosides,4′-S-2′-deoxyribonucleosides and 4′-S-2′-substituted ribonucleosides),bicyclic modified nucleosides (such as bicyclic nucleosides wherein thesugar moiety has a 2′-O—CHR_(a)-4′ bridging group, wherein R_(a) is H,alkyl or substituted alkyl), other modified nucleosides and nucleosideshaving sugar surrogates.

As used herein, “Non-bicyclic modified sugar” or “non-bicyclic modifiedsugar moiety” means a modified sugar moiety that comprises amodification, such as a substituent, that does not form a bridge betweentwo atoms of the sugar to form a second ring.

As used herein, “Linked nucleosides” are nucleosides that are connectedin a continuous sequence (i.e. no additional nucleosides are presentbetween those that are linked).

As used herein, “Mismatch” or “non-complementary” means a nucleobase ofa first oligonucleotide that is not complementary with the correspondingnucleobase of a second oligonucleotide or target nucleic acid when thefirst and second oligomeric compound are aligned.

As used herein, “MOE” means methoxyethoxy. “2′-MOE” means a —OCH₂CH₂OCH₃group at the 2′ position of a furanosyl ring.

As used herein, “Motif” means the pattern of unmodified and/or modifiedsugar moieties, nucleobases, and/or internucleoside linkages, in anoligonucleotide.

As used herein, “Multi-tissue disease or condition” means a disease orcondition affects or is effected by more than one tissue. In treating amulti-tissue disease or condition, it is desirable to affect more thanone tissue type. In certain embodiments, treatment of disease orcondition may be enhanced by treating the disease or condition inmultiple tissues. For example, in certain embodiments, a disease orcondition may manifest itself in the liver tissue and the muscle tissue.In certain embodiments, treating the disease or condition in the livertissue and the muscle tissue will be more effective than treating thedisease in either the liver tissue or the muscle tissue.

As used herein, “Naturally occurring” means found in nature.

As used herein, “Nucleobase” means an unmodified nucleobase or amodified nucleobase. As used herein an “unmodified nucleobase” isadenine (A), thymine (T), cytosine (C), uracil (U), or guanine (G). Asused herein, a “modified nucleobase” is a group of atoms other thanunmodified A, T, C, U, or G capable of pairing with at least oneunmodified nucleobase. Oligomeric compounds are most often preparedhaving nucleobases selected from adenine, guanine, thymine, cytosine,5′-methyl cytosine and uracil. The optionally protected nucleobasescommonly used for the synthesis of oligomeric compounds are6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine,5′-methyl-4-N-benzoylcytosine, thymine and uracil.

As used herein, “Nucleoside” means a compound comprising a nucleobaseand a sugar moiety. The nucleobase and sugar moiety are each,independently, unmodified or modified. As used herein, “modifiednucleoside” means a nucleoside comprising a modified nucleobase and/or amodified sugar moiety. Modified nucleosides include abasic nucleosides,which lack a nucleobase.

As used herein, “Oligomeric compound” means a compound consisting of anoligonucleotide and optionally one or more additional features, such asa conjugate group or other terminal group.

As used herein, “Oligonucleotide” means a strand of linked nucleosidesconnected via internucleoside linkages, wherein each nucleoside andinternucleoside linkage may be modified or unmodified. Unless otherwiseindicated, oligonucleotides consist of 8-50 linked nucleosides. As usedherein, “modified oligonucleotide” means an oligonucleotide, wherein atleast one nucleoside or internucleoside linkage is modified. As usedherein, “unmodified oligonucleotide” means an oligonucleotide that doesnot comprise any nucleoside modifications or internucleosidemodifications.

As used herein, “Pharmaceutically acceptable carrier or diluent” meansany substance suitable for use in administering to an animal. Certainsuch carriers enable pharmaceutical compositions to be formulated as,for example, tablets, pills, dragees, capsules, liquids, gels, syrups,slurries, suspension and lozenges for the oral ingestion by a subject.In certain embodiments, a pharmaceutically acceptable carrier or diluentis sterile water; sterile saline; or sterile buffer solution.

As used herein, “Pharmaceutically acceptable salts” meansphysiologically and pharmaceutically acceptable salts of compounds, suchas oligomeric compounds, i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto.

As used herein, “Pharmaceutical composition” means a mixture ofsubstances suitable for administering to a subject. For example, apharmaceutical composition may comprise an antisense compound and asterile aqueous solution. In certain embodiments, a pharmaceuticalcomposition shows activity in free uptake assay in certain cell lines.

As used herein, “Phosphorus moiety” means a group of atoms comprising aphosphorus atom. In certain embodiments, a phosphorus moiety comprises amono-, di-, or tri-phosphate, or phosphorothioate.

As used herein, “Prodrug” means a therapeutic agent in a form outsidethe body that is converted to a different form within the body or cellsthereof. Typically conversion of a prodrug within the body isfacilitated by the action of an enzymes (e.g., endogenous or viralenzyme) or chemicals present in cells or tissues and/or by physiologicconditions.

As used herein, “RNAi compound” means an antisense compound that acts,at least in part, through RISC or Ago2 to modulate a target nucleic acidand/or protein encoded by a target nucleic acid. RNAi compounds include,but are not limited to double-stranded siRNA, single-stranded RNA(ssRNA), and microRNA, including microRNA mimics. In certainembodiments, an RNAi compound modulates the amount, activity, and/orsplicing of a target nucleic acid. The term RNAi compound excludesantisense oligonucleotides that act through RNase H.

As used herein, “RNA-like nucleoside” means a modified nucleoside otherthan a β-D-ribose nucleoside that provides an A-form (northern) duplexwhen incorporated into an oligomeric compound and duplexed with acomplementary RNA. RNA-like nucleosides are used as replacements for RNAnucleosides in oligomeric compounds to enhance one or more propertiessuch as, for example, nuclease resistance and or hybridization affinity.RNA-like nucleosides include, but are not limited to modified furanosylnucleosides that adopt a 3′-endo conformational geometry when put intoan oligomeric compound. RNA-like nucleosides also include RNA surrogatessuch as F-HNA. RNA-like nucleosides include but are not limited tomodified nucleosides comprising a 2′-substituent group selected from F,O(CH₂)₂OCH₃ (MOE) and OCH₃. RNA-like nucleosides also include but arenot limited to modified nucleosides comprising bicyclic furanosyl sugarmoiety comprising a 4′-CH₂—O-2′, 4′-(CH₂)₂—O-2′, 4′-C(H)[(R)—CH₃]—O-2′or 4′—C(H)[(S)—CH₃]—O-2′ bridging group.

As used herein, “Single-stranded” in reference to an oligomeric compoundmeans such a compound that is not paired with a second oligomericcompound to form a duplex. “Self-complementary” in reference to anoligonucleotide means an oligonucleotide that at least partiallyhybridizes to itself. A compound consisting of one oligomeric compound,wherein the oligonucleotide of the oligomeric compound isself-complementary, is a single-stranded compound. A single-strandedantisense or oligomeric compound may be capable of binding to acomplementary oligomeric compound to form a duplex, in which case itwould no longer be single-stranded.

As used herein, “Sugar moiety” means an unmodified sugar moiety or amodified sugar moiety. As used herein, “unmodified sugar moiety” means a2′-OH(H) furanosyl moiety, as found in RNA (an “unmodified RNA sugarmoiety”), or a 2′-H(H) moiety, as found in DNA (an “unmodified DNA sugarmoiety”). Unmodified sugar moieties have one hydrogen at each of the 1′,3′, and 4′ positions, an oxygen at the 3′ position, and two hydrogens atthe 5′ position. As used herein, “modified sugar moiety” or “modifiedsugar” means a modified furanosyl sugar moiety or a sugar surrogate. Asused herein, modified furanosyl sugar moiety means a furanosyl sugarcomprising a non-hydrogen substituent in place of at least one hydrogenof an unmodified sugar moiety. In certain embodiments, a modifiedfuranosyl sugar moiety is a 2′-substituted sugar moiety. Such modifiedfuranosyl sugar moieties include bicyclic sugars and non-bicyclicsugars. As used herein, “sugar surrogate” or means a modified sugarmoiety having other than a furanosyl moiety that can link a nucleobaseto another group, such as an internucleoside linkage, conjugate group,or terminal group in an oligonucleotide. Modified nucleosides comprisingsugar surrogates can be incorporated into one or more positions withinan oligonucleotide and such oligonucleotides are capable of hybridizingto complementary oligomeric compounds or nucleic acids.

As used herein, “Target nucleic acid” means a naturally occurring,identified nucleic acid. In certain embodiments, target nucleic acidsare endogenous cellular nucleic acids, including, but not limited to RNAtranscripts, pre-mRNA, mRNA, microRNA. In certain embodiments, targetnucleic acids are viral nucleic acids. In certain embodiments, targetnucleic acids are nucleic acids that an antisense compound is designedto affect.

As used herein, “Target region” means a portion of a target nucleic acidto which an antisense compound is designed to hybridize.

In certain embodiments, the oligomeric compounds as provided herein canbe modified by covalent attachment of one or more terminal groups to the5′ or 3′-terminal groups. A terminal group can also be attached at anyother position at one of the terminal ends of the oligomeric compound.As used herein the terms “5′-terminal group”, “3′-terminal group”,“terminal group” and combinations thereof are meant to include usefulgroups known to the art skilled that can be placed on one or both of theterminal ends, including but not limited to the 5′ and 3′-ends of anoligomeric compound respectively, for various purposes such as enablingthe tracking of the oligomeric compound (a fluorescent label or otherreporter group), improving the pharmacokinetics or pharmacodynamics ofthe oligomeric compound (such as for example: uptake and/or delivery) orenhancing one or more other desirable properties of the oligomericcompound (a group for improving nuclease stability or binding affinity).In certain embodiments, 5′ and 3′-terminal groups include withoutlimitation, modified or unmodified nucleosides; two or more linkednucleosides that are independently, modified or unmodified; conjugategroups; capping groups; phosphate moieties; and protecting groups.

I. Certain Oligonucleotides

In certain embodiments, the invention provides oligonucleotides, whichconsist of linked nucleosides. Oligonucleotides may be unmodifiedoligonucleotides (RNA or DNA) or may be modified oligonucleotides.Modified oligonucleotides comprise at least one modification relative tounmodified RNA or DNA (i.e., comprise at least one modified nucleoside(comprising a modified sugar moiety and/or a modified nucleobase) and/orat least one modified internucleoside linkage).

A. Certain Modified Nucleosides

Modified nucleosides comprise a modified sugar moiety or a modifiednucleobase or both a modified sugar moiety and a modified nucleobase.

1. Certain Sugar Moieties

In certain embodiments, modified sugar moieties are non-bicyclicmodified sugar moieties. In certain embodiments, modified sugar moietiesare bicyclic or tricyclic sugar moieties. In certain embodiments,modified sugar moieties are sugar surrogates. Such sugar surrogates maycomprise one or more substitutions corresponding to those of other typesof modified sugar moieties.

In certain embodiments, modified sugar moieties are non-bicyclicmodified sugar moieties comprising a furanosyl ring with one or moreacyclic substituent, including but not limited to substituents at the2′, 4′, and/or 5′ positions. In certain embodiments one or more acyclicsubstituent of non-bicyclic modified sugar moieties is branched.Examples of 2′-substituent groups suitable for non-bicyclic modifiedsugar moieties include but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or“O-methyl”), and 2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments,2′-substituent groups are selected from among: halo, allyl, amino,azido, SH, CN, OCN, CF₃, OCF₃, O—C₁-C₁₀ alkoxy, O—C₁-C₁₀ substitutedalkoxy, O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl, S-alkyl,N(R_(m))-alkyl, O-alkenyl, S-alkenyl, N(R_(m))-alkenyl, O-alkynyl,S-alkynyl, N(R_(m))-alkynyl, O-alkylenyl-O-alkyl, alkynyl, alkaryl,aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃, O(CH₂)₂₀N(R_(m))(R_(n)) orOCH₂C(═O)—N(R_(m))(R_(n)), where each R_(m) and R_(n) is, independently,H, an amino protecting group, or substituted or unsubstituted C₁-C₁₀alkyl, and the 2′-substituent groups described in Cook et al., U.S. Pat.No. 6,531,584; Cook et al., U.S. Pat. No. 5,859,221; and Cook et al.,U.S. Pat. No. 6,005,087. Certain embodiments of these 2′-substituentgroups can be further substituted with one or more substituent groupsindependently selected from among: hydroxyl, amino, alkoxy, carboxy,benzyl, phenyl, nitro (NO₂), thiol, thioalkoxy, thioalkyl, halogen,alkyl, aryl, alkenyl and alkynyl. Examples of 4′-substituent groupssuitable for non-bicyclic modified sugar moieties include but are notlimited to alkoxy (e.g., methoxy), alkyl, and those described inManoharan et al., WO 2015/106128. Examples of 5′-substituent groupssuitable for non-bicyclic modified sugar moieties include but are notlimited to: 5′-methyl (R or S), 5′-vinyl, and 5′-methoxy. In certainembodiments, non-bicyclic modified sugars comprise more than onenon-bridging sugar substituent, for example, 2′-F-5′-methyl sugarmoieties and the modified sugar moieties and modified nucleosidesdescribed in Migawa et al., WO 2008/101157 and Rajeev et al.,US2013/0203836).

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclicmodified nucleoside comprises a sugar moiety comprising a non-bridging2′-substituent group selected from: F, NH₂, N₃, OCF₃, OCH₃, O(CH₂)₃NH₂,CH₂CH═CH₂, OCH₂CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O(CH₂)₂₀N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (OCH₂C(═O)—N(R_(m))(R_(n))), where each R_(m) and R_(n) is,independently, H, an amino protecting group, or substituted orunsubstituted C₁-C₁₀ alkyl.

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclicmodified nucleoside comprises a sugar moiety comprising a non-bridging2′-substituent group selected from: F, OCF₃, OCH₃, OCH₂CH₂OCH₃,O(CH₂)₂SCH₃, O(CH₂)₂₀N(CH₃)₂, O(CH₂)₂O(CH₂)₂N(CH₃)₂, andOCH₂C(═O)—N(H)CH₃ (“NMA”).

In certain embodiments, a 2′-substituted nucleoside or 2′-non-bicyclicmodified nucleoside comprises a sugar moiety comprising a non-bridging2′-substituent group selected from: F, OCH₃, and OCH₂CH₂OCH₃.

Nucleosides comprising modified sugar moieties, such as non-bicyclicmodified sugar moieties, may be referred to by the position(s) of thesubstitution(s) on the sugar moiety of the nucleoside. For example,nucleosides comprising 2′-substituted or 2-modified sugar moieties arereferred to as 2′-substituted nucleosides or 2-modified nucleosides.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. Incertain such embodiments, the bicyclic sugar moiety comprises a bridgebetween the 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′bridging sugar substituents include but are not limited to: 4′-CH₂-2′,4′-(CH₂)₂-2′, 4′-(CH₂)₃-2′, 4′-CH₂—O-2′ (“LNA”), 4′-CH₂—S-2′,4′-(CH₂)₂—O-2′ (“ENA”), 4′-CH(CH₃)—O-2′ (referred to as “constrainedethyl” or “cEt” when in the S configuration), 4′-CH₂—O—CH₂-2′,4′-CH₂—N(R)-2′, 4′-CH(CH₂OCH₃)—O-2′ (“constrained MOE” or “cMOE”) andanalogs thereof (see, e.g., Seth et al., U.S. Pat. No. 7,399,845, Bhatet al., U.S. Pat. No. 7,569,686, Swayze et al., U.S. Pat. No. 7,741,457,and Swayze et al., U.S. Pat. No. 8,022,193), 4′-C(CH₃)(CH₃)—O-2′ andanalogs thereof (see, e.g., Seth et al., U.S. Pat. No. 8,278,283),4′-CH₂—N(OCH₃)-2′ and analogs thereof (see, e.g., Prakash et al., U.S.Pat. No. 8,278,425), 4′-CH₂—O—N(CH₃)-2′ (see, e.g., Allerson et al.,U.S. Pat. No. 7,696,345 and Allerson et al., U.S. Pat. No. 8,124,745),4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Zhou, et al., J. Org. Chem., 2009, 74,118-134), 4′-CH₂—C(═CH₂)-2′ and analogs thereof (see e.g., Seth et al.,U.S. Pat. No. 8,278,426), 4′-C(R_(a)R_(b))—N(R)—O-2′,4′-C(R_(a)R_(b))—O—N(R)-2′, 4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′,wherein each R, R_(a), and R_(b) is, independently, H, a protectinggroup, or C₁-C₁₂ alkyl (see, e.g. Imanishi et al., U.S. Pat. No.7,427,672).

In certain embodiments, such 4′ to 2′ bridges independently comprisefrom 1 to 4 linked groups independently selected from:—[C(R_(a))(R_(b))]_(n)—, —[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a))═C(R_(b))—,—C(R_(a))═N, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—, —Si(R_(a))₂—,—S(═O)_(x)—, and —N(R_(a))—;

wherein:

x is 0, 1, or 2;

n is 1, 2, 3, or 4;

each R_(a) and R_(b) is, independently, H, a protecting group, hydroxyl,C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substitutedC₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl,substituted C₅-C₂₀ aryl, heterocycle radical, substituted heterocycleradical, heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁, N₃,COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)₂-J₁), orsulfoxyl (S(═O)-J₁); and

each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted C₁-C₁₂alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl,substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl, acyl(C(═O)—H), substituted acyl, a heterocycle radical, a substitutedheterocycle radical, C₁-C₁₂ aminoalkyl, substituted C₁-C₁₂ aminoalkyl,or a protecting group.

Additional bicyclic sugar moieties are known in the art, see, forexample: Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443,Albaek et al., J. Org. Chem., 2006, 71, 7731-7740, Singh et al., Chem.Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54,3607-3630; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222;Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al.,J. Am. Chem. Soc., 20017, 129, 8362-8379; Wengel et a., U.S. Pat. No.7,053,207; Imanishi et al., U.S. Pat. No. 6,268,490; Imanishi et al.,U.S. Pat. No. 6,770,748; Imanishi et al., U.S. RE44,779; Wengel et al.,U.S. Pat. No. 6,794,499; Wengel et al., U.S. Pat. No. 6,670,461; Wengelet al., U.S. Pat. No. 7,034,133; Wengel et al., U.S. Pat. No. 8,080,644;Wengel et al., U.S. Pat. No. 8,034,909; Wengel et al., U.S. Pat. No.8,153,365; Wengel et al., U.S. Pat. No. 7,572,582; and Ramasamy et al.,U.S. Pat. No. 6,525,191; Torsten et al., WO 2004/106356; Wengel et al.,WO 1999/014226; Seth et al., WO 2007/134181; Seth et al., U.S. Pat. No.7,547,684; Seth et al., U.S. Pat. No. 7,666,854; Seth et al., U.S. Pat.No. 8,088,746; Seth et al., U.S. Pat. No. 7,750,131; Seth et al., U.S.Pat. No. 8,030,467; Seth et al., U.S. Pat. No. 8,268,980; Seth et al.,U.S. Pat. No. 8,546,556; Seth et al., U.S. Pat. No. 8,530,640; Migawa etal., U.S. Pat. No. 9,012,421; Seth et al., U.S. Pat. No. 8,501,805; andU.S. Patent Publication Nos. Allerson et al., US2008/0039618 and Migawaet al., US2015/0191727.

In certain embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, an LNA nucleoside (describedherein) may be in the α-L configuration or in the β-D configuration.

α-L-methyleneoxy (4′-CH₂—O-2′) or α-L-LNA bicyclic nucleosides have beenincorporated into antisense oligonucleotides that showed antisenseactivity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).Herein, general descriptions of bicyclic nucleosides include bothisomeric configurations. When the positions of specific bicyclicnucleosides (e.g., LNA or cEt) are identified in exemplified embodimentsherein, they are in the β-D configuration, unless otherwise specified.

In certain embodiments, modified sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars).

In certain embodiments, modified sugar moieties are sugar surrogates. Incertain such embodiments, the oxygen atom of the sugar moiety isreplaced, e.g., with a sulfur, carbon or nitrogen atom. In certain suchembodiments, such modified sugar moieties also comprise bridging and/ornon-bridging substituents as described herein. For example, certainsugar surrogates comprise a 4′-sulfur atom and a substitution at the2′-position (see, e.g., Bhat et al., U.S. Pat. No. 7,875,733 and Bhat etal., U.S. Pat. No. 7,939,677) and/or the 5′ position.

In certain embodiments, sugar surrogates comprise rings having otherthan 5 atoms. For example, in certain embodiments, a sugar surrogatecomprises a six-membered tetrahydropyran (“THP”). Such tetrahydropyransmay be further modified or substituted. Nucleosides comprising suchmodified tetrahydropyrans include but are not limited to hexitol nucleicacid (“HNA”), anitol nucleic acid (“ANA”), manitol nucleic acid (“MNA”)(see, e.g., Leumann, CJ. Bioorg. & Med. Chem. 2002, 10, 841-854), fluoroHNA:

(“F-HNA”, see e.g. Swayze et al., U.S. Pat. No. 8,088,904; Swayze etal., U.S. Pat. No. 8,440,803; Swayze et al., U.S. Pat. No. 8,796,437;and Swayze et al., U.S. Pat. No. 9,005,906; F-HNA can also be referredto as a F-THP or 3′-fluoro tetrahydropyran), and nucleosides comprisingadditional modified THP compounds having the formula:

wherein, independently, for each of said modified THP nucleoside:

Bx is a nucleobase moiety;

T₃ and T₄ are each, independently, an internucleoside linking grouplinking the modified THP nucleoside to the remainder of anoligonucleotide or one of T₃ and T₄ is an internucleoside linking grouplinking the modified THP nucleoside to the remainder of anoligonucleotide and the other of T₃ and T₄ is H, a hydroxyl protectinggroup, a linked conjugate group, or a 5′ or 3′-terminal group; q₁, q₂,q₃, q₄, q₅, q₆ and q₇ are each, independently, H, C₁-C₆ alkyl,substituted C₁-C₆ alkyl, C₂-C₆ alkenyl, substituted C₂-C₆ alkenyl, C₂-C₆alkynyl, or substituted C₂-C₆ alkynyl; and each of R₁ and R₂ isindependently selected from among: hydrogen, halogen, substituted orunsubstituted alkoxy, NJ₁J₂, SJ₁, N₃, OC(═X)J₁, OC(═X)NJ₁J₂,NJ₃C(═X)NJ₁J₂, and CN, wherein X is O, S or NJ₁, and each J₁, J₂, and J₃is, independently, H or C₁-C₆ alkyl.

In certain embodiments, modified THP nucleosides are provided whereinq₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certain embodiments, atleast one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other than H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is methyl. Incertain embodiments, modified THP nucleosides are provided wherein oneof R₁ and R₂ is F. In certain embodiments, R₁ is F and R₂ is H, incertain embodiments, R₁ is methoxy and R₂ is H, and in certainembodiments, R₁ is methoxyethoxy and R₂ is H.

In certain embodiments, sugar surrogates comprise rings having more than5 atoms and more than one heteroatom. For example, nucleosidescomprising morpholino sugar moieties and their use in oligonucleotideshave been reported (see, e.g., Braasch et al., Biochemistry, 2002, 41,4503-4510 and Summerton et al., U.S. Pat. No. 5,698,685; Summerton etal., U.S. Pat. No. 5,166,315; Summerton et al., U.S. Pat. No. 5,185,444;and Summerton et al., U.S. Pat. No. 5,034,506). As used here, the term“morpholino” means a sugar surrogate having the following structure:

In certain embodiments, morpholinos may be modified, for example byadding or altering various substituent groups from the above morpholinostructure. Such sugar surrogates are referred to herein as “modifiedmorpholinos.”

In certain embodiments, sugar surrogates comprise acyclic moieties.Examples of nucleosides and oligonucleotides comprising such acyclicsugar surrogates include but are not limited to: peptide nucleic acid(“PNA”), acyclic butyl nucleic acid (see, e.g., Kumar et al., Org.Biomol. Chem., 2013, 11, 5853-5865), and nucleosides andoligonucleotides described in Manoharan et al., WO2011/133876.

Many other bicyclic and tricyclic sugar and sugar surrogate ring systemsare known in the art that can be used in modified nucleosides).

1. Certain Modified Nucleobases

In certain embodiments, modified oligonucleotides comprise one or morenucleoside comprising an unmodified nucleobase. In certain embodiments,modified oligonucleotides comprise one or more nucleoside comprising amodified nucleobase. In certain embodiments, modified oligonucleotidescomprise one or more nucleoside that does not comprise a nucleobase,referred to as an abasic nucleoside.

In certain embodiments, modified nucleobases are selected from:5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynylsubstituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6substituted purines. In certain embodiments, modified nucleobases areselected from: 2-aminopropyladenine, 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N-methyladenine,2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-propynyl (—C≡C—CH₃) uracil, 5-propynylcytosine, 6-azouracil,6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-azaand other 8-substituted purines, 5-halo, particularly 5-bromo,5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine,7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine,7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine,2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5-methyl4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases,hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases. Further modified nucleobases include tricyclicpyrimidines, such as 1,3-diazaphenoxazine-2-one,1,3-diazaphenothiazine-2-one and9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in Merigan et al., U.S. Pat. No. 3,687,808,those disclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859;Englisch et al., Angewandte Chemie, International Edition, 1991, 30,613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications,Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and thosedisclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T.,Ed., CRC Press, 2008, 163-166 and 442-443.

Publications that teach the preparation of certain of the above notedmodified nucleobases as well as other modified nucleobases includewithout limitation, Manoharan et al., US2003/0158403; Manoharan et al.,US2003/0175906; Dinh et al., U.S. Pat. No. 4,845,205; Spielvogel et al.,U.S. Pat. No. 5,130,302; Rogers et al., U.S. Pat. No. 5,134,066;Bischofberger et al., U.S. Pat. No. 5,175,273; Urdea et al., U.S. Pat.No. 5,367,066; Benner et al., U.S. Pat. No. 5,432,272; Matteucci et al.,U.S. Pat. No. 5,434,257; Gmeiner et al., U.S. Pat. No. 5,457,187; Cooket al., U.S. Pat. No. 5,459,255; Froehler et al., U.S. Pat. No.5,484,908; Matteucci et al., U.S. Pat. No. 5,502,177; Hawkins et al.,U.S. Pat. No. 5,525,711; Haralambidis et al., U.S. Pat. No. 5,552,540;Cook et al., U.S. Pat. No. 5,587,469; Froehler et al., U.S. Pat. No.5,594,121; Switzer et al., U.S. Pat. No. 5,596,091; Cook et al., U.S.Pat. No. 5,614,617; Froehler et al., U.S. Pat. No. 5,645,985; Cook etal., U.S. Pat. No. 5,681,941; Cook et al., U.S. Pat. No. 5,811,534; Cooket al., U.S. Pat. No. 5,750,692; Cook et al., U.S. Pat. No. 5,948,903;Cook et al., U.S. Pat. No. 5,587,470; Cook et al., U.S. Pat. No.5,457,191; Matteucci et al., U.S. Pat. No. 5,763,588; Froehler et al.,U.S. Pat. No. 5,830,653; Cook et al., U.S. Pat. No. 5,808,027; Cook etal., U.S. Pat. No. 6,166,199; and Matteucci et al., U.S. Pat. No.6,005,096.

B. Certain Modified Internucleoside Linkages

In certain embodiments, nucleosides of modified oligonucleotides may belinked together using any internucleoside linkage. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus-containinginternucleoside linkages include but are not limited to phosphates,which contain a phosphodiester bond (“P═O”) (also referred to asunmodified or naturally occurring linkages), phosphotriesters,methylphosphonates, phosphoramidates, and phosphorothioates (“P═S”), andphosphorodithioates (“HS—P═S”). Representative non-phosphorus containinginternucleoside linking groups include but are not limited tomethylenemethylimino (—CH₂—N(CH₃)—O—CH₂—), thiodiester, thionocarbamate(—O—C(═O)(NH)—S—); siloxane (—O—SiH₂—O—); and N,N′-dimethylhydrazine(—CH₂—N(CH₃)—N(CH₃)—). Modified internucleoside linkages, compared tonaturally occurring phosphodiester linkages, can be used to alter,typically increase, nuclease resistance of the oligonucleotide. Incertain embodiments, internucleoside linkages having a chiral atom canbe prepared as a racemic mixture, or as separate enantiomers.Representative chiral internucleoside linkages include but are notlimited to alkylphosphonates and phosphorothioates. Methods ofpreparation of phosphorous-containing and non-phosphorous-containinginternucleoside linkages are well known to those skilled in the art.

Neutral internucleoside linkages include, without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′—CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), methoxypropyl, and thioformacetal (3′-S—CH₂—O-5′).Further neutral internucleoside linkages include nonionic linkagescomprising siloxane (dialkylsiloxane), carboxylate ester, carboxamide,sulfide, sulfonate ester and amides (See for example: CarbohydrateModifications in Antisense Research; Y. S. Sanghvi and P. D. Cook, Eds.,ACS Symposium Series 580; Chapters 3 and 4, 40-65). Further neutralinternucleoside linkages include nonionic linkages comprising mixed N,O, S and CH₂ component parts.

C. Certain Motifs

In certain embodiments, modified oligonucleotides comprise one or moremodified nucleoside comprising a modified sugar. In certain embodiments,modified oligonucleotides comprise one or more modified nucleosidescomprising a modified nucleobase. In certain embodiments, modifiedoligonucleotides comprise one or more modified internucleoside linkage.In such embodiments, the modified, unmodified, and differently modifiedsugar moieties, nucleobases, and/or internucleoside linkages of amodified oligonucleotide define a pattern or motif. In certainembodiments, the patterns of sugar moieties, nucleobases, andinternucleoside linkages are each independent of one another. Thus, amodified oligonucleotide may be described by its sugar motif, nucleobasemotif and/or internucleoside linkage motif (as used herein, nucleobasemotif describes the modifications to the nucleobases independent of thesequence of nucleobases).

1. Certain Sugar Motifs

In certain embodiments, oligonucleotides comprise one or more type ofmodified sugar and/or unmodified sugar moiety arranged along theoligonucleotide or region thereof in a defined pattern or sugar motif.In certain instances, such sugar motifs include but are not limited toany of the sugar modifications discussed herein.

In certain embodiments, modified oligonucleotides comprise or consist ofa region having a gapmer motif, which comprises two external regions or“wings” and a central or internal region or “gap.” The three regions ofa gapmer motif (the 5′-wing, the gap, and the 3′-wing) form a contiguoussequence of nucleosides wherein at least some of the sugar moieties ofthe nucleosides of each of the wings differ from at least some of thesugar moieties of the nucleosides of the gap. Specifically, at least thesugar moieties of the nucleosides of each wing that are closest to thegap (the 3′-most nucleoside of the 5′-wing and the 5′-most nucleoside ofthe 3′-wing) differ from the sugar moiety of the neighboring gapnucleosides, thus defining the boundary between the wings and the gap(i.e., the wing/gap junction). In certain embodiments, the sugarmoieties within the gap are the same as one another. In certainembodiments, the gap includes one or more nucleoside having a sugarmoiety that differs from the sugar moiety of one or more othernucleosides of the gap. In certain embodiments, the sugar motifs of thetwo wings are the same as one another (symmetric gapmer). In certainembodiments, the sugar motif of the 5′-wing differs from the sugar motifof the 3′-wing (asymmetric gapmer).

In certain embodiments, the wings of a gapmer comprise 1-5 nucleosides.In certain embodiments, the wings of a gapmer comprise 2-5 nucleosides.In certain embodiments, the wings of a gapmer comprise 3-5 nucleosides.In certain embodiments, the nucleosides of a gapmer are all modifiednucleosides.

In certain embodiments, the gap of a gapmer comprises 7-12 nucleosides.In certain embodiments, the gap of a gapmer comprises 7-10 nucleosides.In certain embodiments, the gap of a gapmer comprises 8-10 nucleosides.In certain embodiments, the gap of a gapmer comprises 10 nucleosides. Incertain embodiment, each nucleoside of the gap of a gapmer is anunmodified 2′-deoxynucleoside.

In certain embodiments, the gapmer is a deoxy gapmer. In suchembodiments, the nucleosides on the gap side of each wing/gap junctionare unmodified 2′-deoxynucleosides and the nucleosides on the wing sidesof each wing/gap junction are modified nucleosides. In certain suchembodiments, each nucleoside of the gap is an unmodified2′-deoxynucleoside. In certain such embodiments, each nucleoside of eachwing is a modified nucleoside.

In certain embodiments, modified oligonucleotides comprise or consist ofa region having a fully modified sugar motif. In such embodiments, eachnucleoside of the fully modified region of the modified oligonucleotidecomprises a modified sugar moiety. In certain such embodiments, eachnucleoside to the entire modified oligonucleotide comprises a modifiedsugar moiety. In certain embodiments, modified oligonucleotides compriseor consist of a region having a fully modified sugar motif, wherein eachnucleoside within the fully modified region comprises the same modifiedsugar moiety, referred to herein as a uniformly modified sugar motif. Incertain embodiments, a fully modified oligonucleotide is a uniformlymodified oligonucleotide. In certain embodiments, each nucleoside of auniformly modified oligonucleotide comprises the same 2′-modification.

2. Certain Nucleobase Motifs

In certain embodiments, oligonucleotides comprise modified and/orunmodified nucleobases arranged along the oligonucleotide or regionthereof in a defined pattern or motif. In certain embodiments, eachnucleobase is modified. In certain embodiments, none of the nucleobasesare modified. In certain embodiments, each purine or each pyrimidine ismodified. In certain embodiments, each adenine is modified. In certainembodiments, each guanine is modified. In certain embodiments, eachthymine is modified. In certain embodiments, each uracil is modified. Incertain embodiments, each cytosine is modified. In certain embodiments,some or all of the cytosine nucleobases in a modified oligonucleotideare 5-methylcytosines.

In certain embodiments, modified oligonucleotides comprise a block ofmodified nucleobases. In certain such embodiments, the block is at the3′-end of the oligonucleotide. In certain embodiments, the block iswithin 3 nucleosides of the 3′-end of the oligonucleotide. In certainembodiments, the block is at the 5′-end of the oligonucleotide. Incertain embodiments, the block is within 3 nucleosides of the 5′-end ofthe oligonucleotide.

In certain embodiments, oligonucleotides having a gapmer motif comprisea nucleoside comprising a modified nucleobase. In certain suchembodiments, one nucleoside comprising a modified nucleobase is in thecentral gap of an oligonucleotide having a gapmer motif. In certain suchembodiments, the sugar moiety of said nucleoside is a 2′-deoxyribosylmoiety. In certain embodiments, the modified nucleobase is selectedfrom: a 2-thiopyrimidine and a 5-propynepyrimidine.

3. Certain Internucleoside Linkage Motifs

In certain embodiments, oligonucleotides comprise modified and/orunmodified internucleoside linkages arranged along the oligonucleotideor region thereof in a defined pattern or motif. In certain embodiments,essentially each internucleoside linking group is a phosphateinternucleoside linkage (P═O). In certain embodiments, eachinternucleoside linking group of a modified oligonucleotide is aphosphorothioate (P═S). In certain embodiments, each internucleosidelinking group of a modified oligonucleotide is independently selectedfrom a phosphorothioate and phosphate internucleoside linkage. Incertain embodiments, the sugar motif of a modified oligonucleotide is agapmer and the internucleoside linkages within the gap are all modified.In certain such embodiments, some or all of the internucleoside linkagesin the wings are phosphodiester linkages. In certain embodiments, theterminal internucleoside linkages are modified.

D. Certain Lengths

In certain embodiments, oligonucleotides (including modifiedoligonucleotides) can have any of a variety of ranges of lengths. Incertain embodiments, oligonucleotides consist of X to Y linkednucleosides, where X represents the fewest number of nucleosides in therange and Y represents the largest number nucleosides in the range. Incertain such embodiments, X and Y are each independently selected from8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, and 50; provided that X≤Y. For example, incertain embodiments, oligonucleotides consist of 12 to 13, 12 to 14, 12to 15, 12 to 16, 12 to 17, 12 to 18, 12 to 19, 12 to 20, 12 to 21, 12 to22, 12 to 23, 12 to 24, 12 to 25, 12 to 26, 12 to 27, 12 to 28, 12 to29, 12 to 30, 13 to 14, 13 to 15, 13 to 16, 13 to 17, 13 to 18, 13 to19, 13 to 20, 13 to 21, 13 to 22, 13 to 23, 13 to 24, 13 to 25, 13 to26, 13 to 27, 13 to 28, 13 to 29, 13 to 30, 14 to 15, 14 to 16, 14 to17, 14 to 18, 14 to 19, 14 to 20, 14 to 21, 14 to 22, 14 to 23, 14 to24, 14 to 25, 14 to 26, 14 to 27, 14 to 28, 14 to 29, 14 to 30, 15 to16, 15 to 17, 15 to 18, 15 to 19, 15 to 20, 15 to 21, 15 to 22, 15 to23, 15 to 24, 15 to 25, 15 to 26, 15 to 27, 15 to 28, 15 to 29, 15 to30, 16 to 17, 16 to 18, 16 to 19, 16 to 20, 16 to 21, 16 to 22, 16 to23, 16 to 24, 16 to 25, 16 to 26, 16 to 27, 16 to 28, 16 to 29, 16 to30, 17 to 18, 17 to 19, 17 to 20, 17 to 21, 17 to 22, 17 to 23, 17 to24, 17 to 25, 17 to 26, 17 to 27, 17 to 28, 17 to 29, 17 to 30, 18 to19, 18 to 20, 18 to 21, 18 to 22, 18 to 23, 18 to 24, 18 to 25, 18 to26, 18 to 27, 18 to 28, 18 to 29, 18 to 30, 19 to 20, 19 to 21, 19 to22, 19 to 23, 19 to 24, 19 to 25, 19 to 26, 19 to 29, 19 to 28, 19 to29, 19 to 30, 20 to 21, 20 to 22, 20 to 23, 20 to 24, 20 to 25, 20 to26, 20 to 27, 20 to 28, 20 to 29, 20 to 30, 21 to 22, 21 to 23, 21 to24, 21 to 25, 21 to 26, 21 to 27, 21 to 28, 21 to 29, 21 to 30, 22 to23, 22 to 24, 22 to 25, 22 to 26, 22 to 27, 22 to 28, 22 to 29, 22 to30, 23 to 24, 23 to 25, 23 to 26, 23 to 27, 23 to 28, 23 to 29, 23 to30, 24 to 25, 24 to 26, 24 to 27, 24 to 28, 24 to 29, 24 to 30, 25 to26, 25 to 27, 25 to 28, 25 to 29, 25 to 30, 26 to 27, 26 to 28, 26 to29, 26 to 30, 27 to 28, 27 to 29, 27 to 30, 28 to 29, 28 to 30, or 29 to30 linked nucleosides

E. Certain Modified Oligonucleotides

In certain embodiments, the above modifications (sugar, nucleobase,internucleoside linkage) are incorporated into a modifiedoligonucleotide. In certain embodiments, modified oligonucleotides arecharacterized by their modification motifs and overall lengths. Incertain embodiments, such parameters are each independent of oneanother. Thus, unless otherwise indicated, each internucleoside linkageof an oligonucleotide having a gapmer sugar motif may be modified orunmodified and may or may not follow the gapmer modification pattern ofthe sugar modifications. For example, the internucleoside linkageswithin the wing regions of a sugar gapmer may be the same or differentfrom one another and may be the same or different from theinternucleoside linkages of the gap region of the sugar motif. Likewise,such sugar gapmer oligonucleotides may comprise one or more modifiednucleobase independent of the gapmer pattern of the sugar modifications.Furthermore, in certain instances, an oligonucleotide is described by anoverall length or range and by lengths or length ranges of two or moreregions (e.g., a region of nucleosides having specified sugarmodifications), in such circumstances it may be possible to selectnumbers for each range that result in an oligonucleotide having anoverall length falling outside the specified range. In suchcircumstances, both elements must be satisfied. For example, in certainembodiments, a modified oligonucleotide consists if of 15-20 linkednucleosides and has a sugar motif consisting of three regions, A, B, andC, wherein region A consists of 2-6 linked nucleosides having aspecified sugar motif, region B consists of 6-10 linked nucleosideshaving a specified sugar motif, and region C consists of 2-6 linkednucleosides having a specified sugar motif. Such embodiments do notinclude modified oligonucleotides where A and C each consist of 6 linkednucleosides and B consists of 10 linked nucleosides (even though thosenumbers of nucleosides are permitted within the requirements for A, B,and C) because the overall length of such oligonucleotide is 22, whichexceeds the upper limit of the overall length of the modifiedoligonucleotide (20). Herein, if a description of an oligonucleotide issilent with respect to one or more parameter, such parameter is notlimited. Thus, a modified oligonucleotide described only as having agapmer sugar motif without further description may have any length,internucleoside linkage motif, and nucleobase motif. Unless otherwiseindicated, all modifications are independent of nucleobase sequence.

F. Nucleobase Sequence

In certain embodiments, oligonucleotides (unmodified or modifiedoligonucleotides) are further described by their nucleobase sequence. Incertain embodiments oligonucleotides have a nucleobase sequence that iscomplementary to a second oligonucleotide or an identified referencenucleic acid, such as a target nucleic acid. In certain suchembodiments, a region of an oligonucleotide has a nucleobase sequencethat is complementary to a second oligonucleotide or an identifiedreference nucleic acid, such as a target nucleic acid. In certainembodiments, the nucleobase sequence of a region or entire length of anoligonucleotide is at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, or 100% complementary to the secondoligonucleotide or nucleic acid, such as a target nucleic acid.

II. Certain Oligomeric Compounds

In certain embodiments, the invention provides oligomeric compounds,which consist of an oligonucleotide (modified or unmodified) andoptionally one or more terminal groups such as a conjugate group.Conjugate groups consist of one or more conjugate group and a conjugatelinking group which links the conjugate group to the oligonucleotide.Conjugate groups may be attached to either or both ends of anoligonucleotide and/or at any internal position. In certain embodiments,conjugate groups are attached to the 2′-position of a nucleoside of amodified oligonucleotide. In certain embodiments, conjugate groups thatare attached to either or both ends of an oligonucleotide are terminalgroups. In certain such embodiments, conjugate groups or terminal groupsare attached at the 3′ and/or 5′-end of oligonucleotides. In certainsuch embodiments, conjugate groups (or other terminal groups) areattached at the 3′-end of oligonucleotides. In certain embodiments,conjugate groups are attached near the 3′-end of oligonucleotides. Incertain embodiments, conjugate groups (or terminal groups) are attachedat the 5′-end of oligonucleotides. In certain embodiments, conjugategroups are attached near the 5′-end of oligonucleotides.

Examples of terminal groups include but are not limited to conjugategroups, capping groups, phosphate moieties, protecting groups, abasicnucleosides, modified or unmodified nucleosides, and two or morenucleosides that are independently modified or unmodified.

A. Certain Conjugate Groups

In certain embodiments, oligonucleotides are covalently attached to oneor more conjugate groups. In certain embodiments, conjugate groupsmodify one or more properties of the attached oligonucleotide, includingbut not limited to pharmacodynamics, pharmacokinetics, stability,binding, absorption, tissue distribution, cellular distribution,cellular uptake, charge and clearance. In certain embodiments, conjugategroups impart a new property on the attached oligonucleotide, e.g.,fluorophores or reporter groups that enable detection of theoligonucleotide. Certain conjugate groups and conjugate moieties havebeen described previously, for example: cholesterol moiety (Letsinger etal., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid(Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4, 1053-1060), athioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad.Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Lett.,1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. AcidsRes., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol orundecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118;Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al.,Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264, 229-237), an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937), a tocopherol group (Nishina et al.,Molecular Therapy Nucleic Acids, 2015, 4, e220; and Nishina et al.,Molecular Therapy, 2008, 16, 734-740), or a GalNAc cluster (e.g.,WO2014/179620).

1. Conjugate Moieties

Conjugate moieties include, without limitation, intercalators, reportermolecules, polyamines, polyamides, peptides, carbohydrates (e.g.,GalNAc), vitamin moieties, polyethylene glycols, thioethers, polyethers,cholesterols, thiocholesterols, cholic acid moieties, folate, lipids,phospholipids, biotin, phenazine, phenanthridine, anthraquinone,adamantane, acridine, fluoresceins, rhodamines, coumarins, fluorophores,and dyes.

In certain embodiments, a conjugate moiety comprises an active drugsubstance, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fen-bufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, fingolimod, flufenamic acid,folinic acid, a benzothiadiazide, chlorothiazide, a diazepine,indo-methicin, a barbiturate, a cephalosporin, a sulfa drug, anantidiabetic, an antibacterial or an antibiotic.

2. Conjugate Linkers

Conjugate moieties are attached to oligonucleotides through conjugatelinkers. In certain oligomeric compounds, the conjugate linker is asingle chemical bond (i.e., the conjugate moiety is attached directly toan oligonucleotide through a single bond). In certain oligomericcompounds, a conjugate moiety is attached to an oligonucleotide via amore complex conjugate linker comprising one or more conjugate linkermoieties, which are sub-units making up a conjugate linker. In certainembodiments, the conjugate linker comprises a chain structure, such as ahydrocarbyl chain, or an oligomer of repeating units such as ethyleneglycol, nucleosides, or amino acid units.

In certain embodiments, a conjugate linker comprises one or more groupsselected from alkyl, amino, oxo, amide, disulfide, polyethylene glycol,ether, thioether, and hydroxylamino. In certain such embodiments, theconjugate linker comprises groups selected from alkyl, amino, oxo, amideand ether groups. In certain embodiments, the conjugate linker comprisesgroups selected from alkyl and amide groups. In certain embodiments, theconjugate linker comprises groups selected from alkyl and ether groups.In certain embodiments, the conjugate linker comprises at least onephosphorus moiety. In certain embodiments, the conjugate linkercomprises at least one phosphate group. In certain embodiments, theconjugate linker includes at least one neutral linking group.

In certain embodiments, conjugate linkers, including the conjugatelinkers described above, are bifunctional linking moieties, e.g., thoseknown in the art to be useful for attaching conjugate groups to parentcompounds, such as the oligonucleotides provided herein. In general, abifunctional linking moiety comprises at least two functional groups.One of the functional groups is selected to bind to a particular site ona parent compound and the other is selected to bind to a conjugategroup. Examples of functional groups used in a bifunctional linkingmoiety include but are not limited to electrophiles for reacting withnucleophilic groups and nucleophiles for reacting with electrophilicgroups. In certain embodiments, bifunctional linking moieties compriseone or more groups selected from amino, hydroxyl, carboxylic acid,thiol, alkyl, alkenyl, and alkynyl.

Examples of conjugate linkers include but are not limited topyrrolidine, 8-amino-3,6-dioxaoctanoic acid (ADO), succinimidyl4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) and6-aminohexanoic acid (AHEX or AHA). Other conjugate linkers include butare not limited to substituted or unsubstituted C₁-C₁₀ alkyl,substituted or unsubstituted C₂-C₁₀ alkenyl or substituted orunsubstituted C₂-C₁₀ alkynyl, wherein a nonlimiting list of preferredsubstituent groups includes hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl andalkynyl.

In certain embodiments, conjugate linkers comprise 1-10linker-nucleosides. In certain embodiments, such linker-nucleosides aremodified nucleosides. In certain embodiments, such linker-nucleosidescomprise a modified sugar moiety. In certain embodiments,linker-nucleosides are unmodified. In certain embodiments,linker-nucleosides comprise an optionally protected heterocyclic baseselected from a purine, substituted purine, pyrimidine or substitutedpyrimidine. In certain embodiments, a cleavable moiety is a nucleosideselected from uracil, thymine, cytosine, 4-N-benzoylcytosine,5-methylcytosine, 4-N-benzoyl-5-methylcytosine, adenine,6-N-benzoyladenine, guanine and 2-N-isobutyrylguanine. In certainembodiments, a cleavable moiety is an unprotectedβ-D-2′-deoxyribonucleoside nucleoside selected from uracil, thymine,cytosine, adenine and guanine. It is typically desirable forlinker-nucleosides to be cleaved from the oligomeric compound after itreaches a target tissue. Accordingly, linker-nucleosides are typicallylinked to one another and to the remainder of the oligomeric compoundthrough cleavable bonds. In certain embodiments, such cleavable bondsare phosphodiester bonds. In certain embodiments, linker nucleosides arelocated at the 5′-terminus of the oligomeric compound. In certainembodiments, linker nucleosides are located at the 3′-terminus of theoligomeric compound.

Herein, linker-nucleosides are not considered to be part of theoligonucleotide. Accordingly, in embodiments in which an oligomericcompound comprises an oligonucleotide consisting of a specified numberor range of linked nucleosides and/or a specified percentcomplementarity to a reference nucleic acid and the oligomeric compoundalso comprises a conjugate group comprising a conjugate linkercomprising linker-nucleosides, those linker-nucleosides are not countedtoward the length of the oligonucleotide and are not used in determiningthe percent complementarity of the oligonucleotide for the referencenucleic acid. For example, an oligomeric compound may comprise (1) amodified oligonucleotide consisting of 8-30 nucleosides and (2) aconjugate group comprising 1-10 linker-nucleosides that are contiguouswith the nucleosides of the modified oligonucleotide. The total numberof contiguous linked nucleosides in such an oligomeric compound is morethan 30. Alternatively, an oligomeric compound may comprise a modifiedoligonucleotide consisting of 8-30 nucleosides and no conjugate group.The total number of contiguous linked nucleosides in such an oligomericcompound is no more than 30. Unless otherwise indicated conjugatelinkers comprise no more than 10 linker-nucleosides. In certainembodiments, conjugate linkers comprise no more than 5linker-nucleosides. In certain embodiments, conjugate linkers compriseno more than 3 linker-nucleosides. In certain embodiments, conjugatelinkers comprise no more than 2 linker-nucleosides. In certainembodiments, conjugate linkers comprise no more than 1linker-nucleoside.

In certain embodiments, it is desirable for a conjugate group to becleaved from the oligonucleotide. For example, in certain circumstancesoligomeric compounds comprising a particular conjugate moiety are bettertaken up by a particular cell type, but once the oligomeric compound hasbeen taken up, it is desirable that the conjugate group be cleaved torelease the unconjugated or parent oligonucleotide. Thus, certainconjugate linkers may comprise one or more cleavable moieties. Incertain embodiments, a cleavable moiety is a cleavable bond. In certainembodiments, a cleavable moiety is a group of atoms comprising at leastone cleavable bond. In certain embodiments, a cleavable moiety comprisesa group of atoms having one, two, three, four, or more than fourcleavable bonds. In certain embodiments, a cleavable moiety isselectively cleaved inside a cell or subcellular compartment, such as alysosome. In certain embodiments, a cleavable moiety is selectivelycleaved by endogenous enzymes, such as nucleases.

In certain embodiments, a cleavable bond is selected from among: anamide, an ester, an ether, one or both esters of a phosphodiester—O—P(═O)(—OH)O—, a phosphate ester —O—P(═O)(—OH)₂, a carbamate,disulfide or a linkage comprising any phosphorus moiety such as—P(═O)(—OH)—. In certain embodiments, a cleavable bond is one or both ofthe esters of a phosphodiester. In certain embodiments, a cleavablemoiety comprises a phosphate or phosphodiester. In certain embodiments,the cleavable moiety is a phosphodiester linkage between anoligonucleotide and a conjugate moiety or conjugate group. In certainembodiments, the cleavable moiety is a phosphodiester linkage between anoligonucleotide and a conjugate linker attaching a conjugate group. Incertain embodiments, the cleavable moiety is a phosphodiester linkagebetween an oligonucleotide and a conjugate group.

In certain embodiments, a cleavable moiety comprises or consists of oneor more linker-nucleosides. In certain such embodiments, the one or morelinker-nucleosides are linked to one another and/or to the remainder ofthe oligomeric compound through cleavable bonds. In certain embodiments,such cleavable bonds are unmodified phosphodiester bonds. In certainembodiments, a cleavable moiety is 2′-deoxy nucleoside that is attachedto either the 3′ or 5′-terminal nucleoside of an oligonucleotide by aphosphate internucleoside linkage and covalently attached to theremainder of the conjugate linker or conjugate moiety by a phosphate orphosphorothioate linkage. In certain such embodiments, the cleavablemoiety is 2′-deoxyadenosine. In certain embodiments, the cleavablemoiety is from one to three nucleosides selected from 2′-deoxyadenosine2′-deoxythymidine and 2′-deoxycytidine.

In certain embodiments, the conjugate group comprises a conjugate linkerincluding a cleavable moiety having one of the formulas:

Wherein the phosphate group attaches the conjugate group to the gappedoligomeric compound. In certain embodiments, the phosphate groupattaches the conjugate group to the 5′-terminal oxygen atom of theoligomeric compound. In certain embodiments, the phosphate groupattaches the conjugate group to the 3′-terminal oxygen atom of theoligomeric compound.

3. Certain Cell-Targeting Conjugate Moieties

In certain embodiments, a conjugate group comprises a cell-targetingconjugate moiety. In certain embodiments, a conjugate group has thegeneral formula:

wherein n is from 1 to about 3, m is 0 when n is 1, m is 1 when n is 2or greater, j is 1 or 0, and k is 1 or 0.

In certain embodiments, n is 1, j is 1 and k is 0. In certainembodiments, n is 1, j is 0 and k is 1. In certain embodiments, n is 1,j is 1 and k is 1. In certain embodiments, n is 2, j is 1 and k is 0.

In certain embodiments, n is 2, j is 0 and k is 1. In certainembodiments, n is 2, j is 1 and k is 1. In certain embodiments, n is 3,j is 1 and k is 0. In certain embodiments, n is 3, j is 0 and k is 1. Incertain embodiments, n is 3, j is 1 and k is 1.

In certain embodiments, conjugate groups comprise cell-targetingmoieties that have at least one tethered ligand. In certain embodiments,cell-targeting moieties comprise two tethered ligands covalentlyattached to a branching group. In certain embodiments, cell-targetingmoieties comprise three tethered ligands covalently attached to abranching group.

In certain embodiments, the cell-targeting moiety comprises a branchinggroup comprising one or more groups selected from alkyl, amino, oxo,amide, disulfide, polyethylene glycol, ether, thioether andhydroxylamino groups. In certain embodiments, the branching groupcomprises a branched aliphatic group comprising groups selected fromalkyl, amino, oxo, amide, disulfide, polyethylene glycol, ether,thioether and hydroxylamino groups. In certain such embodiments, thebranched aliphatic group comprises groups selected from alkyl, amino,oxo, amide and ether groups. In certain such embodiments, the branchedaliphatic group comprises groups selected from alkyl, amino and ethergroups. In certain such embodiments, the branched aliphatic groupcomprises groups selected from alkyl and ether groups. In certainembodiments, the branching group comprises a mono or polycyclic ringsystem.

In certain embodiments, each tether of a cell-targeting moiety comprisesone or more groups selected from alkyl, substituted alkyl, ether,thioether, disulfide, amino, oxo, amide, phosphodiester, andpolyethylene glycol, in any combination. In certain embodiments, eachtether is a linear aliphatic group comprising one or more groupsselected from alkyl, ether, thioether, disulfide, amino, oxo, amide, andpolyethylene glycol, in any combination. In certain embodiments, eachtether is a linear aliphatic group comprising one or more groupsselected from alkyl, phosphodiester, ether, amino, oxo, and amide, inany combination. In certain embodiments, each tether is a linearaliphatic group comprising one or more groups selected from alkyl,ether, amino, oxo, and amide, in any combination. In certainembodiments, each tether is a linear aliphatic group comprising one ormore groups selected from alkyl, amino, and oxo, in any combination. Incertain embodiments, each tether is a linear aliphatic group comprisingone or more groups selected from alkyl, amide and oxo, in anycombination. In certain embodiments, each tether is a linear aliphaticgroup comprising one or more groups selected from alkyl and amide, inany combination. In certain embodiments, each tether is a linearaliphatic group comprising one or more groups selected from alkyl andoxo, in any combination. In certain embodiments, each tether is a linearaliphatic group comprising one or more groups selected from alkyl andphosphodiester, in any combination. In certain embodiments, each tethercomprises at least one phosphorus linking group or neutral linkinggroup. In certain embodiments, each tether comprises a chain from about6 to about 20 atoms in length. In certain embodiments, each tethercomprises a chain from about 10 to about 18 atoms in length. In certainembodiments, each tether comprises about 10 atoms in chain length.

In certain embodiments, each ligand of a cell-targeting moiety has anaffinity for at least one type of receptor on a target cell. In certainembodiments, each ligand has an affinity for at least one type ofreceptor on the surface of a mammalian liver cell. In certainembodiments, each ligand has an affinity for the hepaticasialoglycoprotein receptor (ASGP-R). In certain embodiments, eachligand is a carbohydrate. In certain embodiments, each ligand is,independently selected from galactose, N-acetyl galactoseamine (GalNAc),mannose, glucose, glucoseamine and fucose. In certain embodiments, eachligand is N-acetyl galactoseamine (GalNAc). In certain embodiments, thecell-targeting moiety comprises 3 GalNAc ligands. In certainembodiments, the cell-targeting moiety comprises 2 GalNAc ligands. Incertain embodiments, the cell-targeting moiety comprises 1 GalNAcligand.

In certain embodiments, each ligand of a cell-targeting moiety is acarbohydrate, carbohydrate derivative, modified carbohydrate,polysaccharide, modified polysaccharide, or polysaccharide derivative.In certain such embodiments, the conjugate group comprises acarbohydrate cluster (see, e.g., Maier et al., “Synthesis of AntisenseOligonucleotides Conjugated to a Multivalent Carbohydrate Cluster forCellular Targeting,” Bioconjugate Chemistry, 2003, 14, 18-29 or Rensenet al., “Design and Synthesis of Novel N-Acetylgalactosamine-TerminatedGlycolipids for Targeting of Lipoproteins to the HepaticAsiaglycoprotein Receptor,” J. Med. Chem. 2004, 47, 5798-5808). Incertain such embodiments, each ligand is an amino sugar or a thio sugar.For example, amino sugars may be selected from any number of compoundsknown in the art, such as sialic acid, α-D-galactosamine, β-muramicacid, 2-deoxy-2-methylamino-L-glucopyranose,4,6-dideoxy-4-formamido-2,3-di-O-methyl-D-mannopyranose,2-deoxy-2-sulfoamino-D-glucopyranose and N-sulfo-D-glucosamine, andN-glycoloyl-α-neuraminic acid. For example, thio sugars may be selectedfrom 5-Thio-β-D-glucopyranose, methyl2,3,4-tri-O-acetyl-1-thio-6-O-trityl-α-D-glucopyranoside,4-thio-β-D-galactopyranose, and ethyl3,4,6,7-tetra-O-acetyl-2-deoxy-1,5-dithio-α-D-gluco-heptopyranoside.

In certain embodiments, conjugate groups comprise a cell-targetingmoiety having the formula:

In certain embodiments, conjugate groups comprise a cell-targetingmoiety having the formula:

In certain embodiments, conjugate groups comprise a cell-targetingmoiety having the formula:

In certain embodiments, the conjugate group is attached to the5′position of the oligomeric compound having the formula:

In certain embodiments, the conjugate group is attached to the3′position of the oligomeric compound having the formula:

Representative United States patents, United States patent applicationpublications, international patent application publications, and otherpublications that teach the preparation of certain of the above notedconjugate groups, oligomeric compounds comprising conjugate groups,tethers, conjugate linkers, branching groups, ligands, cleavablemoieties as well as other modifications include without limitation, U.S.Pat. Nos. 5,994,517, 6,300,319, 6,660,720, 6,906,182, 7,262,177,7,491,805, 8,106,022, 7,723,509, US 2006/0148740, US 2011/0123520, WO2013/033230 and WO 2012/037254, Biessen et al., J. Med Chem. 1995, 38,1846-1852, Lee et al., Bioorganic & Medicinal Chemistry 2011, 19,2494-2500, Rensen et al., J. Biol. Chem. 2001, 276, 37577-37584, Rensenet al., J. Med Chem. 2004, 47, 5798-5808, Sliedregt et al., J. Med Chem.1999, 42, 609-618, and Valentijn et al., Tetrahedron, 1997, 53, 759-770.

In certain embodiments, oligomeric compounds comprise modifiedoligonucleotides comprising a gapmer and a conjugate group comprising atleast one, two, or three GalNAc ligands. In certain embodimentsantisense compounds and oligomeric compounds comprise a conjugate groupfound in any of the following references: Lee, Carbohydrate Research,1978, 67, 509-514; Connolly et al., J Biol Chem, 1982, 257, 939-945;Pavia et al., Int J Pep Protein Res, 1983, 22, 539-548; Lee et al.,Biochem, 1984, 23, 4255-4261; Lee et al., Glycoconjugate J, 1987, 4,317-328; Toyokuni et al., Tetrahedron Lett, 1990, 31, 2673-2676; Biessenet al., J Med Chem, 1995, 38, 1538-1546; Valentijn et al., Tetrahedron,1997, 53, 759-770; Kim et al., Tetrahedron Lett, 1997, 38, 3487-3490;Lee et al., Bioconjug Chem, 1997, 8, 762-765; Kato et al., Glycobiol,2001, 11, 821-829; Rensen et al., J Biol Chem, 2001, 276, 37577-37584;Lee et al., Methods Enzymol, 2003, 362, 38-43; Westerlind et al.,Glycoconj J, 2004, 21, 227-241; Lee et al., BioorgMed Chem Lett, 2006,16(19), 5132-5135; Maierhofer et al., BioorgMed Chem, 2007, 15,7661-7676; Khorev et al., BioorgMed Chem, 2008, 16, 5216-5231; Lee etal., BioorgMed Chem, 2011, 19, 2494-2500; Kornilova et al., AnalytBiochem, 2012, 425, 43-46; Pujol et al., Angew Chemie IntEdEngl, 2012,51, 7445-7448; Biessen et al., J Med Chem, 1995, 38, 1846-1852;Sliedregt et al., J Med Chem, 1999, 42, 609-618; Rensen et al., J MedChem, 2004, 47, 5798-5808; Rensen et al., Arterioscler Thromb Vasc Biol,2006, 26, 169-175; van Rossenberg et al., Gene Ther, 2004, 11, 457-464;Sato et al., J Am Chem Soc, 2004, 126, 14013-14022; Lee et al., J OrgChem, 2012, 77, 7564-7571; Biessen et al., FASEB J, 2000, 14, 1784-1792;Rajur et al., Bioconjug Chem, 1997, 8, 935-940; Duff et al., MethodsEnzymol, 2000, 313, 297-321; Maier et al., Bioconjug Chem, 2003, 14,18-29; Jayaprakash et al., Org Lett, 2010, 12, 5410-5413; Manoharan,Antisense Nucleic AcidDrugDev, 2002, 12, 103-128; Merwin et al.,Bioconjug Chem, 1994, 5, 612-620; Tomiya et al., Bioorg Med Chem, 2013,21, 5275-5281; International applications WO1998/013381; WO2011/038356;WO1997/046098; WO2008/098788; WO2004/101619; WO2012/037254;WO2011/120053; WO2011/100131; WO2011/163121; WO2012/177947;WO2013/033230; WO2013/075035; WO2012/083185; WO2012/083046;WO2009/082607; WO2009/134487; WO2010/144740; WO2010/148013;WO1997/020563; WO2010/088537; WO2002/043771; WO2010/129709;WO2012/068187; WO2009/126933; WO2004/024757; WO2010/054406;WO2012/089352; WO2012/089602; WO2013/166121; WO2013/165816; U.S. Pat.Nos. 4,751,219; 8,552,163; 6,908,903; 7,262,177; 5,994,517; 6,300,319;8,106,022; 7,491,805; 7,491,805; 7,582,744; 8,137,695; 6,383,812;6,525,031; 6,660,720; 7,723,509; 8,541,548; 8,344,125; 8,313,772;8,349,308; 8,450,467; 8,501,930; 8,158,601; 7,262,177; 6,906,182;6,620,916; 8,435,491; 8,404,862; 7,851,615; Published U.S. PatentApplication Publications US2011/0097264; US2011/0097265; US2013/0004427;US2005/0164235; US2006/0148740; US2008/0281044; US2010/0240730;US2003/0119724; US2006/0183886; US2008/0206869; US2011/0269814;US2009/0286973; US2011/0207799; US2012/0136042; US2012/0165393;US2008/0281041; US2009/0203135; US2012/0035115; US2012/0095075;US2012/0101148; US2012/0128760; US2012/0157509; US2012/0230938;US2013/0109817; US2013/0121954; US2013/0178512; US2013/0236968;US2011/0123520; US2003/0077829; US2008/0108801; and US2009/0203132.

In certain embodiments, compounds of the invention are single-stranded.In certain embodiments, oligomeric compounds are paired with a secondoligonucleotide or oligomeric compound to form a duplex, which isdouble-stranded.

III. Certain Antisense Compounds

In certain embodiments, the present invention provides antisensecompounds, which comprise or consist of an oligomeric compoundcomprising an antisense oligonucleotide, having a nucleobase sequencescomplementary to that of a target nucleic acid. In certain embodiments,antisense compounds are single-stranded. Such single-stranded antisensecompounds typically comprise or consist of an oligomeric compound thatcomprises or consists of a modified oligonucleotide and optionally aconjugate group. In certain embodiments, antisense compounds aredouble-stranded. Such double-stranded antisense compounds comprise afirst oligomeric compound having a region complementary to a targetnucleic acid and a second oligomeric compound having a regioncomplementary to the first oligomeric compound. The first oligomericcompound of such double stranded antisense compounds typically comprisesor consists of a modified oligonucleotide and optionally a conjugategroup. The oligonucleotide of the second oligomeric compound of suchdouble-stranded antisense compound may be modified or unmodified. Eitheror both oligomeric compounds of a double-stranded antisense compound maycomprise a conjugate group. The oligomeric compounds of double-strandedantisense compounds may include non-complementary overhangingnucleosides.

In certain embodiments, oligomeric compounds of antisense compounds arecapable of hybridizing to a target nucleic acid, resulting in at leastone antisense activity. In certain embodiments, antisense compoundsselectively affect one or more target nucleic acid. Such selectiveantisense compounds comprise a nucleobase sequence that hybridizes toone or more target nucleic acid, resulting in one or more desiredantisense activity and does not hybridize to one or more non-targetnucleic acid or does not hybridize to one or more non-target nucleicacid in such a way that results in significant undesired antisenseactivity.

In certain antisense activities, hybridization of an antisense compoundto a target nucleic acid results in recruitment of a protein thatcleaves the target nucleic acid. For example, certain antisensecompounds result in RNase H mediated cleavage of the target nucleicacid. RNase H is a cellular endonuclease that cleaves the RNA strand ofan RNA:DNA duplex. The DNA in such an RNA:DNA duplex need not beunmodified DNA. In certain embodiments, the invention provides antisensecompounds that are sufficiently “DNA-like” to elicit RNase H activity.Further, in certain embodiments, one or more non-DNA-like nucleoside inthe gap of a gapmer is tolerated.

In certain antisense activities, an antisense compound or a portion ofan antisense compound is loaded into an RNA-induced silencing complex(RISC), ultimately resulting in cleavage of the target nucleic acid. Forexample, certain antisense compounds result in cleavage of the targetnucleic acid by Argonaute. Antisense compounds that are loaded into RISCare RNAi compounds. RNAi compounds may be double-stranded (siRNA) orsingle-stranded (ssRNA).

In certain embodiments, compounds comprising oligonucleotides having agapmer nucleoside motif including one or more modified internucleosidelinkages, having one of formulas I to XVI as described herein, havedesirable properties compared to otherwise equivalent gapmers. Incertain circumstances, it is desirable to identify gapmer motifsresulting in a favorable combination of potent antisense activity andrelatively low toxicity. In certain embodiments, gapped oligomericcompounds of the present invention have a favorable therapeutic index(measure of potency divided by measure of toxicity).

In certain embodiments, hybridization of an antisense compound to atarget nucleic acid does not result in recruitment of a protein thatcleaves that target nucleic acid. In certain such embodiments,hybridization of the antisense compound to the target nucleic acidresults in alteration of splicing of the target nucleic acid. In certainembodiments, hybridization of an antisense compound to a target nucleicacid results in inhibition of a binding interaction between the targetnucleic acid and a protein or other nucleic acid. In certain suchembodiments, hybridization of an antisense compound to a target nucleicacid results in alteration of translation of the target nucleic acid.

Antisense activities may be observed directly or indirectly. In certainembodiments, observation or detection of an antisense activity involvesobservation or detection of a change in an amount of a target nucleicacid or protein encoded by such target nucleic acid, a change in theratio of splice variants of a nucleic acid or protein, and/or aphenotypic change in a cell or animal.

IV. Certain Target Nucleic Acids

In certain embodiments, antisense compounds comprise or consist of anoligonucleotide comprising a region that is complementary to a targetnucleic acid. In certain embodiments, the target nucleic acid is anendogenous RNA molecule. In certain embodiments, the target nucleic acidencodes a protein. In certain such embodiments, the target nucleic acidis selected from: an mRNA and a pre-mRNA, including intronic, exonic anduntranslated regions. In certain embodiments, the target RNA is an mRNA.In certain embodiments, the target nucleic acid is a pre-mRNA. Incertain such embodiments, the target region is entirely within anintron. In certain embodiments, the target region spans an intron/exonjunction. In certain embodiments, the target region is at least 50%within an intron.

In certain embodiments, the target nucleic acid is a non-coding RNA. Incertain such embodiments, the target non-coding RNA is selected from: along-non-coding RNA, a short non-coding RNA, an intronic RNA molecule, asnoRNA, a scaRNA, a microRNA (including pre-microRNA and maturemicroRNA), a ribosomal RNA, and promoter directed RNA. In certainembodiments, the target nucleic acid is a nucleic acid other than amature mRNA. In certain embodiments, the target nucleic acid is anucleic acid other than a mature mRNA or a microRNA. In certainembodiments, the target nucleic acid is a non-coding RNA other than amicroRNA. In certain embodiments, the target nucleic acid is anon-coding RNA other than a microRNA or an intronic region of apre-mRNA. In certain embodiments, the target nucleic acid is a longnon-coding RNA. In certain embodiments, the target nucleic acid is anon-coding RNA associated with splicing of other pre-mRNAs. In certainembodiments, the target nucleic acid is a nuclear-retained non-codingRNA.

In certain embodiments, antisense compounds described herein arecomplementary to a target nucleic acid comprising a single-nucleotidepolymorphism (SNP). In certain such embodiments, the antisense compoundis capable of modulating expression of one allele of the SNP-containingtarget nucleic acid to a greater or lesser extent than it modulatesanother allele. In certain embodiments, an antisense compound hybridizesto a (SNP)-containing target nucleic acid at the single-nucleotidepolymorphism site.

In certain embodiments, antisense compounds are at least partiallycomplementary to more than one target nucleic acid. For example,antisense compounds of the present invention may mimic microRNAs, whichtypically bind to multiple targets.

A. Complementarity/Mismatches to the Target Nucleic Acid

In certain embodiments, antisense compounds comprise antisenseoligonucleotides that are complementary to the target nucleic acid overthe entire length of the oligonucleotide. In certain embodiments, sucholigonucleotides are 99% complementary to the target nucleic acid. Incertain embodiments, such oligonucleotides are 95% complementary to thetarget nucleic acid. In certain embodiments, such oligonucleotides are90% complementary to the target nucleic acid. In certain embodiments,such oligonucleotides are 85% complementary to the target nucleic acid.In certain embodiments, such oligonucleotides are 80% complementary tothe target nucleic acid. In certain embodiments, antisenseoligonucleotides are at least 80% complementary to the target nucleicacid over the entire length of the oligonucleotide and comprise a regionthat is 100% or fully complementary to a target nucleic acid. In certainsuch embodiments, the region of full complementarity is from 6 to 20nucleobases in length. In certain such embodiments, the region of fullcomplementarity is from 10 to 18 nucleobases in length. In certain suchembodiments, the region of full complementarity is from 18 to 20nucleobases in length.

In certain embodiments, the oligomeric compounds of antisense compoundscomprise one or more mismatched nucleobases relative to the targetnucleic acid. In certain such embodiments, antisense activity againstthe target is reduced by such mismatch, but activity against anon-target is reduced by a greater amount. Thus, in certain suchembodiments selectivity of the antisense compound is improved. Incertain embodiments, the mismatch is specifically positioned within anoligonucleotide having a gapmer motif. In certain such embodiments, themismatch is at position 1, 2, 3, 4, 5, 6, 7, or 8 from the 5′-end of thegap region (5′-gap junction). In certain such embodiments, the mismatchis at position 9, 8, 7, 6, 5, 4, 3, 2, 1 from the 3′-end of the gapregion (3a′-gap junction). In certain such embodiments, the mismatch isat position 1, 2, 3, or 4 from the 5′-end of the wing region. In certainsuch embodiments, the mismatch is at position 4, 3, 2, or 1 from the3′-end of the wing region.

B. Certain Target Nucleic Acids in Certain Tissues

In certain embodiments, antisense compounds comprise or consist of anoligonucleotide comprising a region that is complementary to a targetnucleic acid, wherein the target nucleic acid is expressed in anextra-hepatic tissue. Extra-hepatic tissues include, but are not limitedto: skeletal muscle, cardiac muscle, smooth muscle, adipose, whiteadipose, spleen, bone, intestine, adrenal, testes, ovary, pancreas,pituitary, prostate, skin, uterus, bladder, brain, glomerulus, distaltubular epithelium, breast, lung, heart, kidney, ganglion, frontalcortex, spinal cord, trigeminal ganglia, sciatic nerve, dorsal rootganglion, epididymal fat, diaphragm, pancreas, and colon.

V. Certain Pharmaceutical Compositions

In certain embodiments, the present invention provides pharmaceuticalcompositions comprising one or more antisense compound or a saltthereof. In certain such embodiments, the pharmaceutical compositioncomprises a suitable pharmaceutically acceptable diluent or carrier. Incertain embodiments, a pharmaceutical composition comprises a sterilesaline solution and one or more antisense compound. In certainembodiments, such pharmaceutical composition consists of a sterilesaline solution and one or more antisense compound. In certainembodiments, the sterile saline is pharmaceutical grade saline. Incertain embodiments, a pharmaceutical composition comprises one or moreantisense compound and sterile water. In certain embodiments, apharmaceutical composition consists of one antisense compound andsterile water. In certain embodiments, the sterile water ispharmaceutical grade water. In certain embodiments, a pharmaceuticalcomposition comprises one or more antisense compound andphosphate-buffered saline (PBS). In certain embodiments, apharmaceutical composition consists of one or more antisense compoundand sterile PBS. In certain embodiments, the sterile PBS ispharmaceutical grade PBS.

In certain embodiments, pharmaceutical compositions comprise one or moreor antisense compound and one or more excipients. In certain suchembodiments, excipients are selected from water, salt solutions,alcohol, polyethylene glycols, gelatin, lactose, amylase, magnesiumstearate, talc, silicic acid, viscous paraffin, hydroxymethylcelluloseand polyvinylpyrrolidone.

In certain embodiments, antisense compounds may be admixed withpharmaceutically acceptable active and/or inert substances for thepreparation of pharmaceutical compositions or formulations. Compositionsand methods for the formulation of pharmaceutical compositions depend ona number of criteria, including, but not limited to, route ofadministration, extent of disease, or dose to be administered.

In certain embodiments, pharmaceutical compositions comprising anantisense compound encompass any pharmaceutically acceptable salts ofthe antisense compound, esters of the antisense compound, or salts ofsuch esters. In certain embodiments, pharmaceutical compositionscomprising antisense compounds comprising one or more antisenseoligonucleotide, upon administration to an animal, including a human,are capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to pharmaceutically acceptable salts ofantisense compounds, prodrugs, pharmaceutically acceptable salts of suchprodrugs, and other bioequivalents. Suitable pharmaceutically acceptablesalts include, but are not limited to, sodium and potassium salts. Incertain embodiments, prodrugs comprise one or more conjugate groupattached to an oligonucleotide, wherein the conjugate group is cleavedby endogenous nucleases within the body.

Lipid moieties have been used in nucleic acid therapies in a variety ofmethods. In certain such methods, the nucleic acid, such as an antisensecompound, is introduced into preformed liposomes or lipoplexes made ofmixtures of cationic lipids and neutral lipids. In certain methods, DNAcomplexes with mono- or poly-cationic lipids are formed without thepresence of a neutral lipid. In certain embodiments, a lipid moiety isselected to increase distribution of a pharmaceutical agent to aparticular cell or tissue. In certain embodiments, a lipid moiety isselected to increase distribution of a pharmaceutical agent to fattissue. In certain embodiments, a lipid moiety is selected to increasedistribution of a pharmaceutical agent to muscle tissue.

In certain embodiments, pharmaceutical compositions comprise a deliverysystem. Examples of delivery systems include, but are not limited to,liposomes and emulsions. Certain delivery systems are useful forpreparing certain pharmaceutical compositions including those comprisinghydrophobic compounds. In certain embodiments, certain organic solventssuch as dimethylsulfoxide are used.

In certain embodiments, pharmaceutical compositions comprise one or moretissue-specific delivery molecules designed to deliver the one or morepharmaceutical agents of the present invention to specific tissues orcell types. For example, in certain embodiments, pharmaceuticalcompositions include liposomes coated with a tissue-specific antibody.

In certain embodiments, pharmaceutical compositions comprise aco-solvent system. Certain of such co-solvent systems comprise, forexample, benzyl alcohol, a nonpolar surfactant, a water-miscible organicpolymer, and an aqueous phase. In certain embodiments, such co-solventsystems are used for hydrophobic compounds. A non-limiting example ofsuch a co-solvent system is the VPD co-solvent system, which is asolution of absolute ethanol comprising 3% w/v benzyl alcohol, 8% w/v ofthe nonpolar surfactant Polysorbate 80™ and 65% w/v polyethylene glycol300. The proportions of such co-solvent systems may be variedconsiderably without significantly altering their solubility andtoxicity characteristics. Furthermore, the identity of co-solventcomponents may be varied: for example, other surfactants may be usedinstead of Polysorbate 80™; the fraction size of polyethylene glycol maybe varied; other biocompatible polymers may replace polyethylene glycol,e.g., polyvinyl pyrrolidone; and other sugars or polysaccharides maysubstitute for dextrose.

In certain embodiments, pharmaceutical compositions are prepared fororal administration. In certain embodiments, pharmaceutical compositionsare prepared for buccal administration. In certain embodiments, apharmaceutical composition is prepared for administration by injection(e.g., intravenous, subcutaneous, intramuscular, etc.). In certain ofsuch embodiments, a pharmaceutical composition comprises a carrier andis formulated in aqueous solution, such as water or physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiological saline buffer. In certain embodiments, other ingredientsare included (e.g., ingredients that aid in solubility or serve aspreservatives). In certain embodiments, injectable suspensions areprepared using appropriate liquid carriers, suspending agents and thelike. Certain pharmaceutical compositions for injection are presented inunit dosage form, e.g., in ampoules or in multi-dose containers. Certainpharmaceutical compositions for injection are suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents. Certainsolvents suitable for use in pharmaceutical compositions for injectioninclude, but are not limited to, lipophilic solvents and fatty oils,such as sesame oil, synthetic fatty acid esters, such as ethyl oleate ortriglycerides, and liposomes. Aqueous injection suspensions may contain.

Nonlimiting Disclosure and Incorporation by Reference

Each of the literature and patent publications listed herein isincorporated by reference in its entirety. While certain compounds,compositions and methods described herein have been described withspecificity in accordance with certain embodiments, the followingexamples serve only to illustrate the compounds described herein and arenot intended to limit the same. Each of the references, GenBankaccession numbers, and the like recited in the present application isincorporated herein by reference in its entirety.

Although the sequence listing accompanying this filing identifies eachsequence as either “RNA” or “DNA” as required, in reality, thosesequences may be modified with any combination of chemicalmodifications. One of skill in the art will readily appreciate that suchdesignation as “RNA” or “DNA” to describe modified oligonucleotides is,in certain instances, arbitrary. For example, an oligonucleotidecomprising a nucleoside comprising a 2′-OH sugar moiety and a thyminebase could be described as a DNA having a modified sugar (2′-OH in placeof one 2′-H of DNA) or as an RNA having a modified base (thymine(methylated uracil) in place of a uracil of RNA). Accordingly, nucleicacid sequences provided herein, including, but not limited to those inthe sequence listing, are intended to encompass nucleic acids containingany combination of natural or modified RNA and/or DNA, including, butnot limited to such nucleic acids having modified nucleobases. By way offurther example and without limitation, an oligomeric compound havingthe nucleobase sequence “ATCGATCG” encompasses any oligomeric compoundshaving such nucleobase sequence, whether modified or unmodified,including, but not limited to, such compounds comprising RNA bases, suchas those having sequence “AUCGAUCG” and those having some DNA bases andsome RNA bases such as “AUCGATCG” and oligomeric compounds having othermodified nucleobases, such as “AT^(m)CGAUCG,” wherein ^(m)c indicates acytosine base comprising a methyl group at the 5-position.

Certain compounds described herein (e.g., modified oligonucleotides)have one or more asymmetric center and thus give rise to enantiomers,diastereomers, and other stereoisomeric configurations that may bedefined, in terms of absolute stereochemistry, as (R) or (S), as α or βsuch as for sugar anomers, or as (D) or (L), such as for amino acids,etc. Included in the compounds provided herein are all such possibleisomers, including their racemic and optically pure forms, unlessspecified otherwise. Likewise, all cis- and trans-isomers and tautomericforms are also included unless otherwise indicated. Unless otherwiseindicated, compounds described herein are intended to includecorresponding salt forms.

EXAMPLES Example 1 Synthesis of Nucleoside Phosphoramidites

The preparation of nucleoside phosphoramidites is performed followingprocedures that are illustrated herein and in the art such as but notlimited to U.S. Pat. No. 6,426,220 and published PCT WO 02/36743.

Example 2 Synthesis of Oligomeric Compounds

The oligomeric compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as alkylatedderivatives and those having phosphorothioate linkages.

Oligomeric compounds: Unsubstituted and substituted phosphodiester (P═O)oligomeric compounds, including without limitation, oligonucleotides canbe synthesized on an automated DNA synthesizer (Applied Biosystems model394) using standard phosphoramidite chemistry with oxidation by iodine.

In certain embodiments, phosphorothioate internucleoside linkages (P═S)are synthesized similar to phosphodiester internucleoside linkages withthe following exceptions: thiation is effected by utilizing a 10% w/vsolution of 3,H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile forthe oxidation of the phosphite linkages. The thiation reaction step timeis increased to 180 sec and preceded by the normal capping step. Aftercleavage from the CPG column and deblocking in concentrated ammoniumhydroxide at 55° C. (12-16 hr), the oligomeric compounds are recoveredby precipitating with greater than 3 volumes of ethanol from a 1 MNH₄OAc solution. Phosphinate internucleoside linkages can be prepared asdescribed in U.S. Pat. No. 5,508,270.

Alkyl phosphonate internucleoside linkages can be prepared as describedin U.S. Pat. No. 4,469,863.

3′-Deoxy-3′-methylene phosphonate internucleoside linkages can beprepared as described in U.S. Pat. No. 5,610,289 or 5,625,050.

Phosphoramidite internucleoside linkages can be prepared as described inU.S. Pat. No. 5,256,775 or 5,366,878.

Alkylphosphonothioate internucleoside linkages can be prepared asdescribed in published PCT applications PCT/US94/00902 andPCT/US93/06976 (published as WO 94/17093 and WO 94/02499, respectively).

3′-Deoxy-3′-amino phosphoramidate internucleoside linkages can beprepared as described in U.S. Pat. No. 5,476,925.

Phosphotriester internucleoside linkages can be prepared as described inU.S. Pat. No. 5,023,243.

Borano phosphate internucleoside linkages can be prepared as describedin U.S. Pat. Nos. 5,130,302 and 5,177,198.

Oligomeric compounds having one or more non-phosphorus containinginternucleoside linkages including without limitationmethylenemethylimino linked oligonucleosides, also identified as MMIlinked oligonucleosides, methylenedimethylhydrazo linkedoligonucleosides, also identified as MDH linked oligonucleosides,methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligonucleosides, aswell as mixed backbone oligomeric compounds having, for instance,alternating MMI and P═O or P═S linkages can be prepared as described inU.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289.

Formacetal and thioformacetal internucleoside linkages can be preparedas described in U.S. Pat. Nos. 5,264,562 and 5,264,564.

Ethylene oxide internucleoside linkages can be prepared as described inU.S. Pat. No. 5,223,618.

Example 3 Isolation and Purification of Oligomeric Compounds

After cleavage from the controlled pore glass solid support or othersupport medium and deblocking in concentrated ammonium hydroxide at 55°C. for 12-16 hours, the oligomeric compounds, including withoutlimitation oligonucleotides and oligonucleosides, are recovered byprecipitation out of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligomeric compounds are analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresis.The relative amounts of phosphorothioate and phosphodiester linkagesobtained in the synthesis is determined by the ratio of correctmolecular weight relative to the −16 amu product (+/−32+/−48). For somestudies oligomeric compounds are purified by HPLC, as described byChiang et al., J. Biol. Chem. 1991, 266(27), 18162-18171. Resultsobtained with HPLC-purified material are generally similar to thoseobtained with non-HPLC purified material.

Example 4 Synthesis of Oligomeric Compounds Using the 96 Well PlateFormat

Oligomeric compounds, including without limitation oligonucleotides, canbe synthesized via solid phase P(III) phosphoramidite chemistry on anautomated synthesizer capable of assembling 96 sequences simultaneouslyin a 96-well format. Phosphodiester internucleoside linkages areafforded by oxidation with aqueous iodine. Phosphorothioateinternucleoside linkages are generated by sulfurization utilizing3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrousacetonitrile. Standard base-protected beta-cyanoethyl-diiso-propylphosphoramidites can be purchased from commercial vendors (e.g.PE-Applied Biosystems, Foster City, Calif., or Pharmacia, Piscataway,N.J.). Non-standard nucleosides are synthesized as per standard orpatented methods and can be functionalized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligomeric compounds can be cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product is thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 5 Analysis of Oligomeric Compounds Using the 96-Well PlateFormat

The concentration of oligomeric compounds in each well can be assessedby dilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products can be evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE™ MDQ)or, for individually prepared samples, on a commercial CE apparatus(e.g., Beckman P/ACE™ 5000, ABI 270). Base and backbone composition isconfirmed by mass analysis of the oligomeric compounds utilizingelectrospray-mass spectroscopy. All assay test plates are diluted fromthe master plate using single and multi-channel robotic pipettors.Plates are judged to be acceptable if at least 85% of the oligomericcompounds on the plate are at least 85% full length.

Example 6

In Vitro Treatment of Cells with Oligomeric Compounds

The effect of oligomeric compounds on target nucleic acid expression istested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Cell linesderived from multiple tissues and species can be obtained from AmericanType Culture Collection (ATCC, Manassas, Va.).

The following cell type is provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays or RT-PCR.

b.END cells: The mouse brain endothelial cell line b.END was obtainedfrom Dr. Werner Risau at the Max Plank Institute (Bad Nauheim, Germany).b.END cells are routinely cultured in DMEM, high glucose (InvitrogenLife Technologies, Carlsbad, Calif.) supplemented with 10% fetal bovineserum (Invitrogen Life Technologies, Carlsbad, Calif.). Cells areroutinely passaged by trypsinization and dilution when they reachedapproximately 90% confluence. Cells are seeded into 96-well plates(Falcon-Primaria #353872, BD Biosciences, Bedford, Mass.) at a densityof approximately 3000 cells/well for uses including but not limited tooligomeric compound transfection experiments.

Experiments involving treatment of cells with oligomeric compounds:

When cells reach appropriate confluency, they are treated witholigomeric compounds using a transfection method as described.

LIPOFECTIN™

When cells reached 65-75% confluency, they are treated with one or moreoligomeric compounds. The oligomeric compound is mixed with LIPOFECTIN™Invitrogen Life Technologies, Carlsbad, Calif.) in Opti-MEM™-1 reducedserum medium (Invitrogen Life Technologies, Carlsbad, Calif.) to achievethe desired concentration of the oligomeric compound(s) and aLIPOFECTIN™ concentration of 2.5 or 3 μg/mL per 100 nM oligomericcompound(s). This transfection mixture is incubated at room temperaturefor approximately 0.5 hours. For cells grown in 96-well plates, wellsare washed once with 100 μL OPTI-MEM™-1 and then treated with 130 μL ofthe transfection mixture. Cells grown in 24-well plates or otherstandard tissue culture plates are treated similarly, using appropriatevolumes of medium and oligomeric compound(s). Cells are treated and dataare obtained in duplicate or triplicate. After approximately 4-7 hoursof treatment at 37° C., the medium containing the transfection mixtureis replaced with fresh culture medium. Cells are harvested 16-24 hoursafter treatment with oligomeric compound(s).

Other suitable transfection reagents known in the art include, but arenot limited to, CYTOFECTIN™, LIPOFECTAMINE™, OLIGOFECTAMINE™, andFUGENE™. Other suitable transfection methods known in the art include,but are not limited to, electroporation.

Example 7

Real-Time Quantitative PCR Analysis of Target mRNA Levels

Quantitation of target mRNA levels is accomplished by real-timequantitative PCR using the ABI PRISM™ 7600, 7700, or 7900 SequenceDetection System (PE-Applied Biosystems, Foster City, Calif.) accordingto manufacturer's instructions. This is a closed-tube, non-gel-based,fluorescence detection system which allows high-throughput quantitationof polymerase chain reaction (PCR) products in real-time. As opposed tostandard PCR in which amplification products are quantitated after thePCR is completed, products in real-time quantitative PCR are quantitatedas they accumulate. This is accomplished by including in the PCRreaction an oligonucleotide probe that anneals specifically between theforward and reverse PCR primers, and contains two fluorescent dyes. Areporter dye (e.g., FAM or JOE, obtained from either PE-AppliedBiosystems, Foster City, Calif., Operon Technologies Inc., Alameda,Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) isattached to the 5′ end of the probe and a quencher dye (e.g., TAMRA,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 3′ end of the probe. When the probeand dyes are intact, reporter dye emission is quenched by the proximityof the 3′ quencher dye. During amplification, annealing of the probe tothe target sequence creates a substrate that can be cleaved by the5′-exonuclease activity of Taq polymerase. During the extension phase ofthe PCR amplification cycle, cleavage of the probe by Taq polymerasereleases the reporter dye from the remainder of the probe (and hencefrom the quencher moiety) and a sequence-specific fluorescent signal isgenerated. With each cycle, additional reporter dye molecules arecleaved from their respective probes, and the fluorescence intensity ismonitored at regular intervals by laser optics built into the ABI PRISM™Sequence Detection System. In each assay, a series of parallel reactionscontaining serial dilutions of mRNA from untreated control samplesgenerates a standard curve that is used to quantitate the percentinhibition after antisense oligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

RT and PCR reagents are obtained from Invitrogen Life Technologies(Carlsbad, Calif.). RT, real-time PCR is carried out by adding 20 μL PCRcocktail (2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP,dCTP, dCTP and dGTP, 375 nM each of forward primer and reverse primer,125 nM of probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM® Taq, 5Units MuLV reverse transcriptase, and 2.5×ROX dye) to 96-well platescontaining 30 μL total RNA solution (20-200 ng). The RT reaction iscarried out by incubation for 30 minutes at 48° C. Following a 10 minuteincubation at 95° C. to activate the PLATINUM® Taq, 40 cycles of atwo-step PCR protocol are carried out: 95° C. for 15 seconds(denaturation) followed by 60° C. for 1.5 minutes(annealing/-extension).

Gene target quantities obtained by RT, real-time PCR are normalizedusing either the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RIBOGREEN™ (MolecularProbes, Inc. Eugene, Oreg.). GAPDH expression is quantified by real timeRT-PCR, by being run simultaneously with the target, multiplexing, orseparately. Total RNA is quantified using RiboGreen™ RNA quantificationreagent (Molecular Probes, Inc. Eugene, Oreg.). Methods of RNAquantification by RIBOGREEN™ are taught in Jones, L. J., et al,(Analytical Biochemistry, 1998, 265, 368-374).

In this assay, 170 μL of RIBOGREEN™ working reagent (RIBOGREEN™ reagentdiluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA, pH 7.5) is pipetted into a96-well plate containing 30 μL purified, cellular RNA. The plate is readin a CytoFluor 4000 (PE Applied Biosystems) with excitation at 485 nmand emission at 530 nm.

Example 8 Analysis of Inhibition of Target Expression

Antisense modulation of a target expression can be assayed in a varietyof ways known in the art. For example, a target mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR. Real-time quantitative PCR ispresently desired. RNA analysis can be performed on total cellular RNAor poly(A)+ mRNA. One method of RNA analysis of the present disclosureis the use of total cellular RNA as described in other examples herein.Methods of RNA isolation are well known in the art. Northern blotanalysis is also routine in the art. Real-time quantitative (PCR) can beconveniently accomplished using the commercially available ABI PRISM™7600, 7700, or 7900 Sequence Detection System, available from PE-AppliedBiosystems, Foster City, Calif. and used according to manufacturer'sinstructions.

Protein levels of a target can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed to atarget can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art. Methods for preparation ofpolyclonal antisera are taught in, for example, Ausubel, F. M. et al.,Current Protocols in Molecular Biology, Volume 2, pp. 11.12.1-11.12.9,John Wiley & Sons, Inc., 1997. Preparation of monoclonal antibodies istaught in, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons,Inc., 1997.

Immunoprecipitation methods are standard in the art and can be found at,for example, Ausubel, F. M. et al., Current Protocols in MolecularBiology, Volume 2, pp. 10.16.1-10.16.11, John Wiley & Sons, Inc., 1998.Western blot (immunoblot) analysis is standard in the art and can befound at, for example, Ausubel, F. M. et al., Current Protocols inMolecular Biology, Volume 2, pp. 10.8.1-10.8.21, John Wiley & Sons,Inc., 1997. Enzyme-linked immunosorbent assays (ELISA) are standard inthe art and can be found at, for example, Ausubel, F. M. et al., CurrentProtocols in Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley& Sons, Inc., 1991.

Example 9 Design of Phenotypic Assays and In Vivo Studies for the Use ofTarget Inhibitors Phenotypic Assays

Once target inhibitors have been identified by the methods disclosedherein, the oligomeric compounds are further investigated in one or morephenotypic assays, each having measurable endpoints predictive ofefficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of a target in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with atarget inhibitors identified from the in vitro studies as well ascontrol compounds at optimal concentrations which are determined by themethods described above. At the end of the treatment period, treated anduntreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Measurement of the expression of one or more of the genes of the cellafter treatment is also used as an indicator of the efficacy or potencyof the target inhibitors. Hallmark genes, or those genes suspected to beassociated with a specific disease state, condition, or phenotype, aremeasured in both treated and untreated cells.

In Vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

Example 10 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA is isolated according to Miura et al., (Clin. Chem., 1996,42, 1758-1764). Other methods for poly(A)+ mRNA isolation are routine inthe art. Briefly, for cells grown on 96-well plates, growth medium isremoved from the cells and each well is washed with 200 μL cold PBS. 60μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5%NP-40, 20 mM vanadyl-ribonucleoside complex) is added to each well, theplate is gently agitated and then incubated at room temperature for fiveminutes. 55 μL of lysate is transferred to Oligo d(T) coated 96-wellplates (AGCT Inc., Irvine Calif.). Plates are incubated for 60 minutesat room temperature, washed 3 times with 200 μL of wash buffer (10 mMTris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After the final wash, the plateis blotted on paper towels to remove excess wash buffer and thenair-dried for 5 minutes. 60 μL of elution buffer (5 mM Tris-HCl pH 7.6),preheated to 70° C., is added to each well, the plate is incubated on a90° C. hot plate for 5 minutes, and the eluate is then transferred to afresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA is isolated using an RNEASY 96™ kit and buffers purchased fromQiagen Inc. (Valencia, Calif.) following the manufacturer's recommendedprocedures. Briefly, for cells grown on 96-well plates, growth medium isremoved from the cells and each well is washed with 200 μL cold PBS. 150μL Buffer RLT is added to each well and the plate vigorously agitatedfor 20 seconds. 150 μL of 70% ethanol is then added to each well and thecontents mixed by pipetting three times up and down. The samples arethen transferred to the RNEASY 96™ well plate attached to a QIAVAC™manifold fitted with a waste collection tray and attached to a vacuumsource. Vacuum is applied for 1 minute. 500 μL of Buffer RW1 is added toeach well of the RNEASY 96™ plate and incubated for 15 minutes and thevacuum is again applied for 1 minute. An additional 500 μL of Buffer RW1is added to each well of the RNEASY 96™ plate and the vacuum is appliedfor 2 minutes. 1 mL of Buffer RPE is then added to each well of theRNEASY 96™ plate and the vacuum applied for a period of 90 seconds. TheBuffer RPE wash is then repeated and the vacuum is applied for anadditional 3 minutes. The plate is then removed from the QIAVAC™manifold and blotted dry on paper towels. The plate is then re-attachedto the QIAVAC™ manifold fitted with a collection tube rack containing1.2 mL collection tubes. RNA is then eluted by pipetting 140 μL of RNAsefree water into each well, incubating 1 minute, and then applying thevacuum for 3 minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 11 Target-Specific Primers and Probes

Probes and primers may be designed to hybridize to a target sequence,using published sequence information.

For example, for human PTEN, the following primer-probe set was designedusing published sequence information (GENBANK™ accession numberU92436.1, SEQ ID NO: 1).

Forward primer: (SEQ ID NO: 2) AATGGCTAAGTGAAGATGACAATCAT.Reverse primer: (SEQ ID NO: 3) TGCACATATCATTACACCAGTTCGT.And the PCR probe:

FAM-TTGCAGCAATTCACTGTAAAGCTGGAAAGG-TAMRA (SEQ ID NO: 4), where FAM isthe fluorescent dye and TAMRA is the quencher dye.

Example 12 Western Blot Analysis of Target Protein Levels

Western blot analysis (immunoblot analysis) is carried out usingstandard methods. Cells are harvested 16-20 h after oligonucleotidetreatment, washed once with PBS, suspended in Laemmli buffer (100μL/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel. Gelsare run for 1.5 hours at 150 V, and transferred to membrane for westernblotting. Appropriate primary antibody directed to a target is used,with a radiolabeled or fluorescently labeled secondary antibody directedagainst the primary antibody species. Bands are visualized using aPHOSPHORIMAGER™ (Molecular Dynamics, Sunnyvale Calif.).

Example 13 General Procedure for Synthesis of DMT Diamidite MorpholinoMonomer, Compound 4

Wherein Bx is an optionally protected heterocyclic base moiety.

Compound 4 is prepared from commercially available compounds 1 and 3 asper published literature procedures (Caruthers et al., J Am Chem Soc,2016, 138(48), 15663-15672).

Following these procedures Compound 4a (Bx=thymine) was prepared. ¹H NMRwas consistent with the structure.

Following these procedures compounds 4b-d are prepared (Compound 4b,Bx=5′-methyl-4-N-isobutyrylcytosine; Compound 4c, Bx=6-N-benzoyladenine;and Compound 4d, Bx=2-N-isobutyrylguanine).

Example 14 General Procedure for Synthesis of Compound 5

Compound 5 was prepared by suspending Mg (s, 4.18 g) in dry THF (150 mL)and 1-bromo-3-methoxypropane (12 mL) was added in portions over 20minutes with periodic cooling in an ice bath to keep the temperatureunder 30° C. The reaction was then allowed to stir an additional 10minutes at room temperature. The solution was then added to a secondflask containing bis(diisopropylamino)chlorophosphine (25 grams) as astirred cold suspension in dry diethyl ether. The reaction was allowedto come to room temperature and stirred for one hour. The reactionmixture was filtered through a plug of Celite, and the solids are rinsedwith Et₂O. The filtrates are pooled, and the solvent were removed. Theresidue was suspended in dry ACN (75 ml) and transferred to a separatoryfunnel. The product was extracted with hexanes washed with additionalACN and concentrated to provide Compound 5, 27.10 g (84.1% yield) whichwas used in the next reaction immediately. ¹³P NMR was consistent withthe structures.

Example 15 General Procedure for Synthesis of DMT Diamidite MorpholinoMonomer Compound 6, Synthesis of Compounds 6a-d

Compound 2 (1.84 mmoles, prepared as illustrated in Example 13) istreated with tetrazole (200 mg, 2.76 mmol, 1.5 equiv), 1-methylimidazole (75 μL, 0.92 mmol, 0.5 equiv) and Compound 5 (1.5 equivalents,840 mg, prepared as illustrated in Example 14) in dry DMF (18 mL) for 20hours at room temperature under nitrogen. The reaction was diluted withEtOAc (50 ml) and washed with 1:1 H₂O and saturated NaHCO₃ (2×50 ml)followed by brine (1×50 ml). The organic layers are pooled, dried overMgSO₄, filtered, and concentrated. The crude material is purified bysilica gel chromatography to give the final compound.

Following these procedures Compound 6a (Bx=thymine) was prepared in a50% yield. ¹H NMR was consistent with the structure.

Example 16 General Procedure for Synthesis of DMT Diamidite MorpholinoMonomer Compound 8, Synthesis of Compounds 8a-d

Compound 2 (18.9 mmoles, prepared as illustrated in Example 13) istreated with tetrazole (1.06 g), 1-methyl imidazole (665 μL) andCompound 7 (1.5 equivalents, commercially available from ChemGenes) inDMF (190 mL) for 20 hours at which time the solvent was removed underreduced pressure with the temperature maintained below 35° C. Theresidue is then diluted with EtOAc and washed with 1:1 H₂O and brinefollowed by, H₂O, saturated NaHCO₃ and NaCl (sat). The organic layersare pooled, dried over MgSO₄, filtered, and concentrated. The crudematerial is purified by silica gel chromatography to give the finalcompound.

Following these procedures compounds 8a-d are prepared (Compound 8a,Bx=thymine; Compound 8b, Bx=5′-methyl-4-N-isobutyrylcytosine; Compound8c, Bx=6-N-benzoyladenine; and Compound 8d, Bx=2-N-isobutyrylguanine).

Example 17 Morpholino Oligonucleotide Synthesis

Morpholino modified oligonucleotides were synthesized on a 2 μmol scaleon an ABI 394 DNA/RNA synthesizer using polystyrene-based VIMADUnylinker™ support. Fully protected nucleoside phosphoramidites wereincorporated using standard solid-phase oligonucleotide synthesis, i.e.3% dichloroacetic acid in DCM for deblocking, 1 M 4,5-dicyanoimidazole0.1 M N-methylimidazole in acetonitrile as activator for amiditecouplings, acetic anhydride in THF and 10% 1-methylimidazole inTHF/pyridine for capping, 0.02 M iodine in pyridine:water 9:1 (v:v) foroxidation and 0.1 M xanthene hydride in pyridine:acetonitrile 1:1 (v:v)for sulfurization. DNA and (S)-cEt building blocks were dissolved inacetonitrile (0.1 M) and incorporated using 2×4 min coupling time whilemorpholino diamidites (Compound 4 wherein R is thymine, prepared as perthe procedure of Example 13) were dissolved in acetonitrile at 0.15 Mand coupled for 2×6 min. At the end of the synthesis, the 5′ DMT groupwas removed on support and cyanoethyl protecting groups removed usingtriethylamine:acetonitrile 1:1 (v:v). The remaining protecting groupswere cleaved in concentrated aqueous ammonia at 55° C. for 8 hrs. Thecrude oligonucleotides were purified by ion-exchange-HPLC using a lineargradient of buffer A and B. Buffer A: 50 mM NaHCO₃ in acetonitrile:water3:7 (v:v), buffer B: 1.5 M NaBr, 50 mM NaHCO₃ in acetonitrile:water 3:7(v:v). The purified oligonucleotides were desalted using C18reverse-phase cartridges.

Example 18 Thermal Stability Assay

A series of modified oligonucleotides were evaluated in a thermalstability (T_(m)) assay. A Cary 100 Bio spectrophotometer with the CaryWin UV Thermal program was used to measure absorbance vs. temperature.For the T_(m) experiments, oligonucleotides were prepared at aconcentration of 8 μM in a buffer of 100 mM Na+, 10 mM phosphate and 0.1mM EDTA (pH 7). The concentration of the oligonucleotides was determinedat 85° C. The concentration of each oligonucleotide was 4 μM aftermixing of equal volumes of test oligonucleotide and complimentary RNAstrand. Oligonucleotides were hybridized with the complimentary RNAstrand by heating the duplex to 90° C. for 5 minutes followed by coolingto room temperature. Using the spectrophotometer, T_(m) measurementswere taken by heating the duplex solution at a rate of 0.5° C./min incuvette starting @ 15° C. and heating to 85° C. T_(m) values weredetermined using Vant Hoff calculations (A₂₆₀ vs temperature curve)using non self-complementary sequences where the minimum absorbancewhich relates to the duplex and the maximum absorbance which relates tothe non-duplex single strand are manually integrated into the program.The oligonucleotides were hybridized to complementary phosphodiesterlinked DNA and or RNA as listed below.

In Table 1, 3/10/3 cEt gapmer oligonucleotides either unmodified or with1 morpholino monomer located at position 4, 6, 7 or 9 were hybridized tocomplementary RNA for Tm measurement. All of the internucleosidelinkages are phosphorothioate except that morpholino monomers are linkedfrom the morpholino ring nitrogen by a phosphoramidate to the adjacentDNA monomer. The internucleoside linkages in the complementary RNA arephosphodiesters.

In Table 2, 12mer oligonucleotides that are either unmodified, have 3morpholino monomers alternating with DNA monomers, or have a continuoussequence of 6 morpholino monomers which are hybridized to complementaryRNA for Tm measurement. The alternating and continuous motifs werelocated internally in 12mers flanked on each side by 3 or 4 DNAmonomers. Each of the internucleoside linkages are phosphodiester exceptthat each morpholino monomer is linked from the morpholino ring nitrogenby a phosphoramidate to the adjacent DNA monomer at the 5′ position. Theinternucleoside linkages in the complementary RNA are phosphodiesters.

The cEt and morpholino monomers are illustrated below. The results arepresented in tables 1 and 2 below.

TABLE 1 SEQ ID NO./ Tm/ ISIS #/*ION# Composition (5′ to 3′) ΔTm ° C.Morpholino mods 05/792775 UAAUGUGAGAACAUGC n/a RNA compliment, PO06/558807 G_(k) ^(m)C_(k)A_(k)TGTT^(m)CT^(m)CA^(m)CAT_(k)T_(k)A_(k)64.9/0.0 unmodified, PS 06/*1044689 G_(k) ^(m)C_(k)A _(k) T_(o)GTT^(m)CT^(m)CA^(m)CAT_(k)T_(k)A_(k) 56.7/−8.2morpholino at pos 4, PS 06/*1044690 G_(k) ^(m)C_(k)A_(k)TG T_(o)T^(m)CT^(m)CA^(m)CAT_(k)T_(k)A_(k) 66.2/1.3 morpholino at pos 6, PS06/*1044691 G_(k) ^(m)C_(k)A_(k)TGT T _(o)^(m)CT^(m)CA^(m)CAT_(k)T_(k)A_(k) 67.4/2.5 morpholino at pos 7, PS06/*1044692 G_(k) ^(m)C_(k)A_(k)TGTT^(m)C T _(o)^(m)CA^(m)CAT_(k)T_(k)A_(k) 65.4/0.5 morpholino at pos 9, PS

TABLE 2 SEQ ID Tm/ΔTm ° C. Morpholino NO./ISIS Composition Tm/ΔTm ° C.period mods/ #/*ION# (5′ to 3′) (against DNA) (against RNA) linkages PO08/438705 GCGTTTTTTGCT 50.6/0.0 45.6/0.0 DNA 08/*1051474 GCG T _(o)T T_(o)T T _(o)TGCT 59.1/2.8 56.5/3.6 alternating 08/*1051473 GCG T _(o) T_(o) T _(o) T _(o) T _(o) T _(o)GCT 49.0/0.6 continuous 07/483795AGCAAAAAACGC n/a n/a DNA compl. 07/438704 AGCAAAAAACGC n/a n/aRNA compl.

Nucleosides followed by a subscript “k” are (S)-cEt modifiednucleosides, “T” indicates a morpholino monomer and all other monomersare β-D-2′-deoxyribonucleosides, “^(m)C” indicates a 5-methyl cytidineand subscript and italicized “o” indicates a 3′-phosphoramidateinternucleoside linkage. The remainder of the internucleoside linkagesin the oligonucleotide backbone are listed in the table as being “PS”phosphorothioate or “PO” phosphodiester.

Wherein Bx is a heterocyclic base.

Example 19 Modified Oligonucleotides Targeting CXCL12 In Vitro Study

Modified oligonucleotides were designed based on the controloligonucleotide ISIS 558807, as illustrated in Example 18. The 3/10/3gapmer design was modified by incorporation of a single morpholinomonomer at various positions within the gap region. Two morpholinomodified oligonucleotides is prepared for each modified position tocompare the effect of having a phosphate versus a thiophosphate linkageattaching the morpholino monomer to the 3′-adjacentβ-D-2′-deoxy-ribonucleotide. The resulting modified oligonucleotideswere tested for their ability to inhibit CXCL12 (Chemokine ligand 12)and Raptor mRNA expression levels. The potency of the modifiedoligonucleotides was evaluated and compared to the controloligonucleotide.

The oligonucleotides were tested in vitro in mouse b.END cells byelectroporation. Cells at a density of 20,000 cells per well weretransfected using electroporation with 0.027, 0.082, 0.25, 0.74, 2.22,6.67 and 20 uM concentrations of each of the oligonucleotides listedbelow. After a treatment period of approximately 24 hours, RNA wasisolated from the cells and mRNA levels were measured by quantitativereal-time PCR and the CXCL12 mRNA and Raptor mRNA levels were adjustedaccording to total RNA content, as measured by RIBOGREEN®.

The half maximal inhibitory concentration (IC₅₀) of each oligonucleotidelisted above was calculated by plotting the concentration ofoligonucleotide versus the percent inhibition of CXCL12 mRNA or RaptormRNA expression achieved at each concentration, and noting theconcentration of oligonucleotide at which 50% inhibition of CXCL12 mRNAexpression is achieved compared to the control. The results arepresented in Table 3 below.

TABLE 3 SEQ CXCL12 RAPTOR ID NO./ISIS IC50 IC50 #/*ION#Composition (5′ to 3′) (nM) (nM) morpholino linkage 06/558807 G_(k)^(m)C_(k)A_(k)TGTT^(m)CT^(m)CA^(m)CAT_(k)T_(k)A_(k)  47    800 Control06/*1044689 G_(k) ^(m)C_(k)A _(k) T _(o)GTT^(m)CT^(m)CA^(m)CAT_(k)T_(k)A_(k) 405 >20000 phosphoramidate06/*1048416 G_(k) ^(m)C_(k)A _(k) T _(s)GTT^(m)CT^(m)CA^(m)CAT_(k)T_(k)A_(k) 333 >20000 thiophosphoramidate06/*1044690 G_(k) ^(m)C_(k)A_(k)TG T _(o)T^(m)CT^(m)CA^(m)CAT_(k)T_(k)A_(k) 182   4100 phosphoramidate06/*1048417 G_(k) ^(m)C_(k)A_(k)TG T _(s)T^(m)CT^(m)CA^(m)CAT_(k)T_(k)A_(k) 159   3300 thiophosphoramidate06/*1044691 G_(k) ^(m)C_(k)A_(k)TGT T _(o)^(m)CT^(m)CA^(m)CAT_(k)T_(k)A_(k) 128   4400 phosphoramidate 06/*1048418G_(k) ^(m)C_(k)A_(k)TGT T _(s) ^(m)CT^(m)CA^(m)CAT_(k)T_(k)A_(k) 134  5200 thiophosphoramidate 06/*1044692 G_(k) ^(m)C_(k)A_(k)TGTT^(m)C T_(o) ^(m)CA^(m)CAT_(k)T_(k)A_(k) 145   1900 phosphoramidate 06/*1048419G_(k) ^(m)C_(k)A_(k)TGTT^(m)C T _(s) ^(m)CA^(m)CAT_(k)T_(k)A_(k) 119  1100 thiophosphoramidate

Nucleosides followed by a subscript “k” are (S)-cEt modifiednucleosides, “T” indicates a morpholino monomer and all other monomersare β-D-2′-deoxyribonucleosides, “^(m)C” indicates a 5-methyl cytidine,subscript and italicized “o” indicates a 3′-phosphoramidateinternucleoside linkage and subscript and italicized “s” indicates a3′-thiophosphoramidate internucleoside linkage. All internucleosidelinkages are phosphorothioate internucleoside linkages.

1. A compound having Formula I:

wherein: Bx is an optionally protected heterocyclic base moiety; T₁ is Hor a hydroxyl protecting group; and R₁ is C₁₋₆ alkyl or substituted C₁₋₆alkyl comprising an optionally protected substituent group selected fromhalogen, OJ₁, NJ₁J₂, SJ₁, N₃, OC(=Q)J₁, OC(=Q)NJ₁J₂, NJ₃C(=Q)NJ₁J₂ andCN, wherein each J₁, J₂, and J₃ is, independently, H or C₁-C₆ alkyl, andQ is O, S, or NJ₁.
 2. The compound of claim 1, wherein Bx is uracil,thymine, cytosine, 4-N-benzoylcytosine, 4-N-isobutyrylcytosine,5′-methyl cytosine, 5′-methyl-4-N-benzoylcytosine,5′-methyl-4-N-isbutyrylcytosine, adenine, 6-N-benzoyladenine, guanine,or 2-N-isobutyrylguanine.
 3. (canceled)
 4. The compound of claim 1,wherein T₁ is 4,4′-dimethoxytrityl.
 5. (canceled)
 6. The compound ofclaim 1, wherein R₁ is methyl or —(CH₂)₃—OCH₃. 7.-9. (canceled)
 10. Anoligomeric compound comprising from 10 to 30 monomer subunits wherein atleast one monomer subunit has Formula III:

wherein independently for each monomer subunit having Formula III: Bx isan optionally protected heterocyclic base moiety; T₃ is hydroxyl, aprotected hydroxyl, an internucleoside linking group linking the monomersubunit of Formula III to the 5′-remainder of the oligomeric compound,or a terminal group; T₅ is H, C₁₋₆ alkyl, a terminal group, or anitrogen protecting group when located on the 3′-terminus of theoligomeric compound, wherein at least one T₅ is a linking group havingthe formula:

wherein independently for each T₅: R₂ is hydroxyl, C₁₋₆ alkyl, orsubstituted C₁₋₆ alkyl comprising an optionally protected substituentgroup selected from halogen, OJ₁, NJ₁J₂, SJ₁, N₃, OC(=Q)J₁, OC(=Q)NJ₁J₂,NJ₃C(=Q)NJ₁J₂, and CN, wherein each J₁, J₂, and J₃ is, independently, Hor C₁-C₆ alkyl, and Q is O, S, or NJ₁; X is O or S, wherein when X is O,at least one R₂ is other than hydroxyl; and T₄ is a single bondconnecting said linking group to the 3′-remainder of the oligomericcompound or T₄ forms a single bond with a T₃ from a second monomersubunit having Formula III.
 11. (canceled)
 12. The oligomeric compoundof claim 10, wherein each Bx is, independently, uracil, thymine,cytosine, 4-N-benzoylcytosine, 4-N-isobutyrylcytosine, 5′-methylcytosine, 5′-methyl-4-N-benzoylcytosine,5′-methyl-4-N-isbutyrylcytosine, adenine, 6-N-benzoyladenine, guanine,or 2-N-isobutyrylguanine.
 13. (canceled)
 14. The oligomeric compound ofclaim 10, wherein: (a) at least one T₃ is an internucleoside linkinggroup linking the monomer subunit of Formula III to the 5′-remainder ofthe oligomeric compound; and/or (b) one T₃ is hydroxyl or one T₃ is aterminal group. 15.-17. (canceled)
 18. The oligomeric compound of claim10, wherein one T₅ is a methyl, one T₅ is 4,4′-dimethoxytrityl, or oneT₅ is a terminal group. 19.-20. (canceled)
 21. The oligomeric compoundof claim 10, wherein at least one T₅ is said linking group. 22.-27.(canceled)
 28. The oligomeric compound of claim 10, wherein each X is O.29. (canceled)
 30. The oligomeric compound of claim 10, wherein each R₂is hydroxyl, methyl, or —(CH₂)₃—OCH₃. 31.-38. (canceled)
 39. Theoligomeric compound of claim 10, comprising a gapped oligomeric compoundhaving a first region consisting of from 2 to 5 monomer subunits, asecond region consisting of from 2 to 5 monomer subunits, and a gapregion located between the first and second region consisting of from 8to 12 monomer subunits, wherein each monomer subunit in the first andsecond regions is a modified nucleoside and each monomer subunit in thegap region is an unmodified nucleoside or a modified nucleosidedifferent from the modified nucleosides in the first and second regionsand wherein at least one of the monomer subunits has Formula III. 40.(canceled)
 41. The oligomeric compound of claim 39, wherein each monomersubunit in the gap region is a β-D-2′-deoxyribonucleoside.
 42. Theoligomeric compound of claim 39, comprising one monomer subunit havingFormula III in the gap region. 43.-44. (canceled)
 45. The oligomericcompound of claim 39, comprising at least one monomer subunit havingFormula III in one of the first and second regions. 46.-52. (canceled)53. The oligomeric compound of claim 39, wherein each modifiednucleoside in the first and second region comprises a modified sugarmoiety. 54.-57. (canceled)
 58. The oligomeric compound of claim 39,wherein at least one modified nucleoside in the first and second regioncomprises a sugar surrogate. 59.-60. (canceled)
 61. An oligomericcompound comprising from 12 to 28 monomer subunits comprising at leastone T(Z_(a)T_(b))_(c)Z_(d) motif, where each a and b is, independently,from 1 to about 8, c is from 1 to about 12, d is 0 or 1, one of T and Zis a β-D-2′-deoxyribonucleoside and the other of T and Z is a monomersubunit having Formula III:

wherein independently for each monomer subunit having Formula III: Bx isan optionally protected heterocyclic base moiety; T₃ is hydroxyl, aprotected hydroxyl, an internucleoside linking group linking the monomersubunit of Formula III to the 5′-remainder of the oligomeric compound,or a terminal group; T₅ is H, C₁₋₆ alkyl, a nitrogen protecting, or alinking group having the formula:

wherein independently for each T₅: R₂ is hydroxyl, C₁₋₆ alkyl, orsubstituted C₁₋₆ alkyl comprising an optionally protected substituentgroup selected from halogen, OJ₁, NJ₁J₂, SJ₁, N₃, OC(=Q)J₁, OC(=Q)NJ₁J₂,NJ₃C(=Q)NJ₁J₂, and CN, wherein each J₁, J₂, and J₃ is, independently, Hor C₁-C₆ alkyl, and Q is O, S, or NJ₁; X is O or S; and T₄ is a singlebond connecting said linking group to the 3′-remainder of the oligomericcompound or T₄ forms a single bond with a T₃ from a second monomersubunit having Formula III.
 62. (canceled)
 63. The oligomeric compoundof claim 61, wherein each Bx is, independently, uracil, thymine,cytosine, 5-methylcytosine, adenine or guanine.
 64. The oligomericcompound of claim 61, wherein each X is O.
 65. (canceled)
 66. Theoligomeric compound of claim 61, wherein each R₂ is hydroxyl, methyl, or—(CH₂)₃—OCH₃. 67.-73. (canceled)
 74. The oligomeric compound of claim61, comprising one T(Z_(a)T_(b))_(c)Z_(d) motif. 75.-77. (canceled) 78.The oligomeric compound of claim 61, wherein each a and b is,independently, from 1 to
 3. 79.-80. (canceled)
 81. The oligomericcompound of claim 61, wherein c is 1, 2, or
 3. 82.-84. (canceled) 85.The oligomeric compound of claim 61, wherein d is 0 or
 1. 86.-90.(canceled)
 91. The oligomeric compound of claim 61, wherein theoligomeric compound comprises at least from 1 to 3 3′-terminalβ-D-2′-deoxyribonucleoside and/or at least from 1 to 3 5′-terminal13-D-2′-deoxyribonucleosides. 92.-97. (canceled)
 98. A method ofinhibiting gene expression comprising contacting one or more cells, atissue, or an animal with an oligomeric compound of claim 10, whereinsaid oligomeric compound is complementary to a target RNA. 99.-104.(canceled)
 105. A method of inhibiting gene expression comprisingcontacting one or more cells, a tissue, or an animal with an oligomericcompound of claim 61, wherein said oligomeric compound is complementaryto a target RNA.